CAN 11885
No. of Pages 10, Model 5G
17 May 2014 Cancer Letters xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet 5 6
Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition
3 4 7 8 9
10 1 5 2 2 13 14 15 16 17 18 19 20 21 22 23 24
Q1
Ranjita Das a, Kaushik Bhattacharya a, Suman K. Samanta a, Bikas C. Pal b, Chitra Mandal a,⇑ a b
Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, Kolkata, India National Institute of Pharmaceutical Education and Research, Kolkata, India
a r t i c l e
i n f o
Article history: Received 8 January 2014 Received in revised form 11 April 2014 Accepted 1 May 2014 Available online xxxx Keywords: Mahanine Cisplatin Cervical cancer Synergism STAT3
a b s t r a c t Toxicity reduction of cisplatin is necessary for improved treatment of cancer. Here we have demonstrated the synergistic growth-inhibitory effect of cisplatin on cervical cancer cells in-combination with a nontoxic herbal carbazole alkaloid, mahanine. Mahanine enhanced cisplatin-induced apoptosis and reduced its effective concentration 5–8 folds. Mahanine inhibited JAK1 and Src and subsequently promoted proteasome-mediated degradation of STAT3. This event was further enhanced in-combination with cisplatin and subsequently inhibited cancer cell migration. Collectively, our results revealed that mahanine may be a prospective agent to reduce the concentration of cisplatin in adjunct for the treatment of cancer and thereby decreasing its toxicity. Ó 2014 Published by Elsevier Ireland Ltd.
26 27 28 29 30 31 32 33 34 35
36 37 38
Introduction
39
Cervical cancer is the third most common malignancy worldwide and it comprises of 13% of all cancers [1]. Human papilloma virus (HPV) infection with high risk type is the main factor for the development of cervical cancer. Among 15 subtypes of highrisk HPV strains, types 16 and 18 are mainly responsible for 76.7% of this cancer in Indian women [2]. Recently, two prophylactic HPV vaccines (Gardasil and Cervarix) are in the market. However, they have no benefit over HPV infection or cervical cancer [3]. Thus, new strategies are still needed. Current treatments are radiotherapy, chemotherapy and surgery. Cisplatin is the drug of choice either alone or in combination with topotecan. However, severe side-effects like bone-marrow depression, neutropenia, thrombocytopenia and anaemia due to haematological toxicity along with nephrotoxicity and neurotoxicity [4] and acquired chemoresistance [5] throughout the course of treatment have limited the usage of cisplatin. To conquer the restrictions in clinical use of cisplatin, combined therapy with dietary compounds is recommended [6–8]. Signal Transducer and Activator of Transcription 3 (STAT3) plays an important role in cellular survival, proliferation, and
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
⇑ Corresponding author. Address: Cancer Biology and Inflammatory Disorders Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India. Tel.: +91 33 2429 8861; fax: +91 33 2473 5197. E-mail addresses: [email protected], [email protected] (C. Mandal).
differentiation. Constitutively activated STAT3 is found in breast, prostrate, lung, ovarian, head and neck, leukemia, lymphoma and cervical cancers etc. [9–10]. Upstream activators of STAT3 include epidermal growth factor receptor (EGFR), IL-6 receptors or nonreceptor tyrosine kinases, Src or Janus kinase (JAK). Phosphorylation at tyrosine (Tyr) residue 705 activates STAT3 resulting in its dimerization, nuclear translocation, DNA binding and transcriptional activation of target genes. Activated STAT3 acts as a proto-oncogene by regulating proliferation and apoptosisassociated proteins such as cyclin D1, c-myc, survivin, Bcl-xL, Bcl-2 and Fas. STAT3 also increases invasion and metastasis by inducing matrix metalloproteinases (MMPs) and angiogenesis by the activation of VEGF and HIF-1a [11]. Overexpressed STAT3 confers resistance to cisplatin-induced apoptosis in cancers [12]. Therefore, inhibition of STAT3 activation may possibly sensitize cancer cells to cisplatin-induced apoptosis [13]. Mahanine, a carbazole alkaloid, purified from well-known Indian herbal plant Murraya koenigii, induced apoptosis in histiocytic lymphoma, promylocytic leukemia and prostate cancer cells [14,15]. In leukemic cells, mahanine induced apoptosis through mitochondrial death cascade and Fas-FasL activation [16,17]. In pancreatic cancer, it inhibited Hsp90 through reactive oxygen species (ROS) [18]. C-7-OH and 9-NH are two functional groups of mahanine. They were responsible for its cytotoxicity and minor groove binding with DNA [19]. It reduced in vivo xenograft [17] and orthotopic [18] tumours and showed nontoxicity to total body mass of normal Balb/c and athymic nude mice [15]. It activated
http://dx.doi.org/10.1016/j.canlet.2014.05.005 0304-3835/Ó 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 2 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106
R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
epigenetically suppressed tumour suppressor gene RASSF1A [20]. Recent study reported that mahanine and 5-fluorouracil combination exert synergistic increase in colon cancer cell death coupled with activation of tumour suppressor proteins, PTEN and p53/ p73 [21]. Hence, we wanted to explore this as a candidate for adjunct therapy with cisplatin in cervical cancer. Here, we have investigated that mahanine exerts anti-proliferative activity in cervical cancer cells alone. It potentially induced apoptosis in combination with cisplatin and reduced the concentration of this toxic drug by 5–8 folds thereby synergistically enhances its effect. The combination-induced apoptosis involved activation of caspase and mitochondria. Furthermore, mahanine potentially deactivated STAT3 by suppressing its phosphorylation. The decreased expression of JAK1 and Src might play important role in deactivation of STAT3. Mahanine also promoted ubiquitindependent proteasome-mediated p-STAT3 degradation. In combination with cisplatin, mahanine enhanced further inhibition of STAT3 phosphorylation, its upstream JAK1 and Src and cancer cell migration. Thus, mahanine could be a potent and valuable chemotherapeutic agent to reduce the toxicity of cisplatin by decreasing its definite concentration.
where (DX)1 and (DX)2 were the concentrations of each drug alone to exert x% effect and (D)1 and (D)2 were the drugs in combination to elicit the same effect. CI < 1, = 1 and > 1 indicate synergism, additivity and antagonism respectively. We further evaluated the fold decrease of each drug as a result of synergism represented as drugreduction index (DRI).
150 151 152 153 154
Apoptosis assay
155
Externalizations of phosphatidylserine were verified by double staining the cells with FITC-annexin V and PI [21]. Briefly, both cells (1 106) were treated either with cisplatin (3 lM) and mahanine (12–20 lM) alone or in combination for 24 h. Next, washed cells were resuspended in annexin-V binding buffer, kept for 45 min at 25 °C followed by incubation with FITC-annexin V and PI (5 lg/ml) for 20 min at 4 °C in dark. Cells were acquired and analysed with CellQuest Pro software on a FACSCalibur flow cytometer (BD).
156 157 158 159 160 161 162
Measurement of mitochondrial membrane potential
163
6
Both cells (1 10 ) were exposed to cisplatin (3 lM) and mahanine (12 lM) alone or their combination for 24 h, washed and incubated with JC1 (25 lM) for 30 min in dark at 37 °C. Cells were analysed by FACS to determine mitochondrial transmembrane depolarization [17] which was represented as mean fluorescence intensity (MFI) of green fluorescence.
164 165 166 167 168
Subcellular fractionation
169 6
107
Materials and methods
108
Reagents
109 110 111 112 113 114 115 116 117 118 119 120 121
The primary antibodies against phospho-STAT3 (Tyr705), STAT3, phosphoJAK1, JAK1, phopho-c-myc, c-myc, cyclin D1, MMP9, Bak, Bid, Bcl-xl, Fas, Caspase-8, Caspase-9, b-actin, poly-ADP ribose polymerase (PARP) and horseradish peroxidase (HRP)-linked secondary antibodies were from Cell Signalling Technology, USA. Primary antibody HDAC-3, FITC-annexin V and Mitoscreen kit (JC1) were from BD Bioscience (San Diego, USA). IMDM cell culture medium, FCS, antibiotic–antimycotic, and trypsin–EDTA were from Invitrogen, USA. Cisplatin, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI), molecular grade BSA, protein A-Sepharose 4B, Tween-20 and dimethyl sulphoxide (DMSO), Src Inhibitor-1 and cucurbitacin B hydrate (JAK inhibitor) were from Sigma–Aldrich, USA. NE-PER nuclear and cytoplasmic extraction reagents and SuperSignal West Pico imaging system were from Thermo-scientific, USA.
122
Mahanine purification and its characterization
123 124 125 126
Mahanine was purified as described earlier [14] from fresh leaves of an Indian plant, M. koenigii (Rutaceae family). The purity was confirmed by HPLC and LC-MS. 1 [H] and 13[C] NMR spectral data analyses established its structure as mahanine (Fig. 1A).
127
Cell lines and cell culture
128 129 130 131
Cervical cancer cell lines, HeLa (HPV-18) and SiHa (HPV-16) were obtained from American Type Culture Collection (Manassas, VA). The cells were grown in IMDM supplemented with 10% FCS and 1% antibiotic antimycotic mixture in a humidified incubator at 37 °C containing 5% CO2.
132
Cytotoxicity assay
133 134 135 136 137 138 139
The cytotoxicity of mahanine and cisplatin were assessed by MTT assay as described previously [18]. Briefly, HeLa and SiHa (1 104/well) cells were treated with mahanine (0–24 lM) and cisplatin (0–20 lM) for 24–48 h. MTT (0.4 mg/ml) was added and further incubated for 3 h in dark at 37 °C. The generated formazan complexes were dissolved in DMSO and the absorbance was measured at 550 nm with spectrophotometer (Thermo). Cell viability was derived from percentage of MTT conversion in treated relative to untreated cells defined as 100%.
140
Combination study with cisplatin
141 142 143 144 145 146
HeLa and SiHa (1 104) cells were treated with cisplatin (0–6 lM) and mahanine (2–24 lM) alone or in combination at a fixed concentration ratio of 1:4 (cisplatin:mahanine). The inhibitory effect was assessed after 48 h by MTT assay and evaluated based on combination index (CI). It was calculated using Chou–Talalay equation by Calcusyn software version 2.1 (Biosoft) [21,22] using the following equation:
147 149
CI ¼ ðDÞ1 =ðDX Þ1 þ ðDÞ2 =ðDX Þ2
Both cells (1 10 ) were treated with cisplatin (3 lM) and mahanine (0–16 lM) or with their combination for 24 h. Treated cells were separated into cytosol and nuclear fractions by NE-PERÒ kit following manufacturer’s protocol. Briefly, washed cells were resuspended in cytosol extraction reagent, vortexed, centrifuged and the supernatant was collected as the cytosolic fraction. The pellet was incubated with nuclear extraction reagent, centrifuged and supernatant used as the nuclear fraction.
170 171 172 173 174 175 176
Western blot analysis
177
Both cells (1 106) were exposed to cisplatin (3 lM) and mahanine (0–16 lM) or combination for 24 h. The cells were washed, resuspended in PBS, sonicated (2 watt and 3 pulses) and followed by cold centrifugation at 10,000 g [23]. The supernatants were used as cell lysates for further experiments. Equal amounts of protein (50 lg) were electrophorsed on SDS–PAGE (7.5–12%) and electrotransferred onto nitrocellulose membranes. The membranes were blocked with TBS-2% BSA and probed with appropriate primary antibodies at 4 °C for overnight. The blots were washed with TBS containing 0.1% Tween-20 and processed using HRP-secondary antibodies. Signals were detected with the West-pico ECL system.
178 179 180 181 182 183 184 185 186
Immunoprecipitation
187
For the detection of proteasome-dependent ubiquitination of p-STAT3 (Tyr 705), SiHa cells (1 106) were pretreated with MG132 (10 lM) for 1 h followed by mahanine treatment (12 lM) for 24 h. Total cell lysates were prepared. Proteins (300 lg) were incubated with anti-p-STAT3 antibody (1:100) for overnight at 4 °C and further with Protein A-Sepharose 4B. The immune complex was resolved by SDS–PAGE (7.5%) and detected using anti-ubiquitin antibody.
188 189 190 191 192 193
Scratch-Wound assay
194
Both cells (1 106) were grown to >90% confluency and starved in reducedserum (0.5%) media for overnight. Next, three separate linear scratches were made by a 200 ll micropipette tip. Wounded monolayers were washed thrice to remove cell debris, kept in 10% FBS medium and incubated with cisplatin (3 lM) and mahanine (12 lM) alone or their combination for 24 h. Images were taken using phasecontrast microscopy.
195 196 197 198 199 200
Statistical analysis
201
The data were obtained from at least three independent experiments. All statistical analyses were performed by Microsoft Excel and GraphPad Prism and evaluated by paired t test. The results were represented as mean ±SD from independent experiments. P values 1 indicates antagonism. The CI values < 1 indicated that interaction between cisplatin (2–5 lM) and mahanine (8–20 lM) was synergistic in both HeLa and SiHa cells.
257 258 259 260 261 262 263 264 265 266 267 268 269
also showed more PARP and Bid cleavage compared to mahanine while cisplatin could not induce these cleavages (Fig. 3B). Involvement of mitochondria is reported in mahanine-induced apoptosis which is a prooxidant molecule [17] and moreover ROS accumulation in mitochondrial lumen leads to the alteration of mitochondrial inner membrane potential [24]. This prompted us to measure mitochondrial membrane potential. Combination resulted in enhanced membrane depolarization signified by the higher MFI of green fluorescence in both cells (Fig. 4). Taken together, these suggested that the combination induces potential apoptosis in cervical cancer cells. Moreover, caspase activation and loss of mitochondrial transmembrane potential attributed to this apoptosis.
270
Mahanine suppressed STAT3 activation
271
Constitutive activation of STAT3 signalling is reported in cervical carcinoma [25] and inhibition of this may play a role in cancer interference [26]. In this context, we investigated whether mahanine can inhibit the STAT3 activation in these cells. Mahanine significantly inhibited phosphorylation of STAT3 (Tyr705) in a concentration-dependent manner. Besides, the total protein levels of STAT3 were almost unchanged, however, decreased only at 16 lM of mahanine (Fig. 5A). Src and JAK tyrosine kinases contribute in the activation of STAT3 [11]. To further elucidate mahanine-mediated suppression of p-STAT3, expression of total and phosphorylated Src and JAK1 proteins were checked. Mahanine resulted in a reduction of phosphorylation of Src and JAK1 in a concentration-dependent manner. The total Src expression was also decreased after mahanine treatment. Though, no significant inhibition was observed in total JAK1 (Fig. 5A). Tyr705 phosphorylation activates STAT3, promotes its dimerization and nuclear localization [9]. We found that mahanine
272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288
reduces the levels of total and phosphorylated STAT3 in nucleus whereas it was increased in cytosol (Fig. 5B). Therefore, we suggested that mahanine induces inhibition of STAT3 signalling in cervical cancer.
289
Mahanine promoted ubiquitin-dependent proteasome-mediated degradation of p-STAT3 (Tyr705)
293
Turnover of p-STAT3 is reported to be regulated by the ubiquitin-dependent proteasomal degradation [27]. Therefore, next we investigated any involvement of ubiquitin-dependent degradation in p-STAT3. MG132 pre-treated HeLa and SiHa cells restored both phosphorylated and total STAT3 (Fig. 5C). Furthermore, enhanced polyubiquitination of p-STAT3 was observed in the presence of MG132 in SiHa cells (Fig. 5D). These findings suggested that mahanine mediates ubiquitination of p-STAT3 leading to its reduction in cervical cancer cells.
295
Cisplatin in-combination with mahanine enhanced deactivation of STAT3 and its downstream proteins
304
Over expression of STAT3 is reported to confer resistance in cisplatin-induced apoptosis [28]. By now, we demonstrated that mahanine suppresses STAT3 activation. Therefore, here we wanted to check combination-induced STAT3 regulation in this cancer. Total STAT3 and p-STAT3 status in both cells remained almost unaffected after cisplatin (3 lM) treatment (Fig. 6A). Similarly, mahanine (12 lM) alone also had little effect on total STAT3 and p-STAT3 in HeLa whereas reduction was observed in SiHa cells. However, combination-exposure considerably decreased phosphorylation of STAT3 in both cells (Fig. 6A). Additionally, cisplatin alone was unable to reduce total and phosphorylation of Src and JAK1 whereas mahanine could suppress these proteins. More importantly, combination-treatment enhanced the reduction of p-Src and p-JAK1. Similar inhibition was observed in presence of their respective inhibitors (Fig. S-6 A and B). We also checked the p-STAT3 level in cisplatin-treated cells along with the cells treated with Src and JAK inhibitors separately and found that both inhibitors inhibits p-STAT3 level whereas cisplatin at 3 lM concentration was unable to reduce the level of p-STAT3. Moreover, combination treatment blocked nuclear translocation of p-STAT3 more than when used them individually, while in cytosol this treatment resulted in the accumulation of p-STAT3 (Fig. 6B). STAT3 activation promotes tumorigenesis by regulating the expression of various gene products involved in cell survival, proliferation and angiogenesis [11]. Accordingly, we wanted to ensure the status of a few downstream proteins of STAT3 after combination treatment. The combination-exposure enhanced the decrease of total and phosphorylated c-myc and c-Jun both in HeLa and SiHa cells than single agent. In contrast, c-myc and c-Jun were almost unchanged in single agent treatment (Figs. 6C and S-3). Moreover, among the angiogenic factors regulated by STAT3, we checked VEGF expression which was also decreased significantly after combination treatment (Fig. S-2). Altogether these results suggested
306
Table 3 Dose reduction index (DRI) values for cisplatin in combination with mahanine. Cell lines
Drug:compound
Molar ratio (cisplatin:mahanine)
DRI (75% fraction affected level)
HeLa SiHa
Cisplatin:mahanine Cisplatin:mahanine
1:4 1:4
4.991 8.369
The DRI signifies the fold decrease of cisplatin as a result of synergistic combination in compared with the concentration of a single agent needed to achieve the same effect. Concentrations of cisplatin were reduced 5-folds in HeLa and 8-folds in SiHa to achieve a 75% inhibition of cell proliferation respectively when the cells were exposed to combination treatment at fixed molar ratios after 48 h.
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
290 291 292
294
296 297 298 299 300 301 302 303
305
307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 6
R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
Fig. 3. Mahanine and cisplatin combination induced more apoptosis in cervical cancer cells. (A) HeLa and SiHa cells were exposed to either cisplatin (3 lM) or mahanine (12 lM) alone or in combination at ratio of 1:4 (cisplatin:mahanine) for 24 h. Increased apoptosis by the combination treatment compared to single agent alone was assessed by FITC-annexin-V and PI positivity through flow cytometry. (B) Both cell lines were treated with either cisplatin (4 lM) or mahanine (16 lM) alone or their combination at 1:4 (cisplatin:mahanine) for 24 h. Treated cells were sonicated to prepare cell lysates, electrophorsed and analysed by Western blot with anti-caspase 8, 9, Bid, PARP antibodies as described in Materials and Methods. Each experiment was done at least three times.
340 341
enhancement of STAT3 deactivation in combination-treated cervical cancer cells.
342
Combination of mahanine and cisplatin decreased more cell motility
343
352
MMPs play a pivotal role in cancer cell motility and invasion among which, MMP-9 is the transcriptional targets of STAT3 [11]. In this perspective, we examined the status of MMP-9 and demonstrated more decreased expression of it in the combination-treated cells (Fig. 7A). Furthermore, scratch-wound assay showed additional decreased motility of both cells after combination-treatment as indicated by unchanged area of wound. Combination treated cells were migrated little compared to single agent (Fig. 7B). These data assured that cervical cancer cell motility is inhibited more when cisplatin was used in combination with mahanine.
353
Discussion
354
The main objective of our study was to reduce the concentration of known anticancer drug (cisplatin) widely used for treat-
344 345 346 347 348 349 350 351
355
ment of different cancers. Accordingly, we have used a nontoxic herbal carbazole alkaloid, mahanine along with cisplatin. Here, we have used cervical cancer as a model system. The main achievement of our study is to establish mahanine as a synergistic modulator of cisplatin in cervical cancer. Mahanine and cisplatin alone exerted growth-inhibitory activity against these cancer cells. The importance of this study is that it synergistically augmented the cytotoxicity of cisplatin even at 5–8-fold reduced concentration. Mahanine alone inhibited STAT3 activation whereas; cisplatin under identical condition had little effect on STAT3 deactivation. However, mahanine in-combination with cisplatin, at the same dose, enhanced inhibition of STAT3 activation, augmented proteasome-mediated degradation and reduced migration of cervical cancer cells compared to single agent treatment. Our study gives us a new hope to reduce the concentration of a well known anti-cancer drug (cisplatin). Severe toxicity and acquired drug resistance are associated with cisplatin treatment [5]. Combination of such toxic chemotherapeutics with nontoxic naturally occurring compounds having anticancer properties would be new strategy to overcome this problem [21]. We demonstrated earlier that mahanine exhibits
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
7
Fig. 4. Mahanine and cisplatin combination induced additional depolarization of mitochondrial membrane potential in cervical cancer. Cells were exposed to either cisplatin (3 lM) or mahanine (12 lM) alone or their combination at ratio of 1:4 (cisplatin:mahanine). After incubation, cells were washed stained with JC-1 and analysed by flow cytometry. Mitochondrial transmembrane depolarization was measured by the increased MFI of green fluorescence in combination treatment than in single agent alone.
377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404
anti-proliferative activity against several cancers both in vitro and in vivo [15–18]. More importantly, it was nontoxic towards normal tissues and total body mass of treated mice [15]. It also synergistically reduced the concentration of 5-fluorouracil for apoptosis of colon cancer cells [21]. Here, we demonstrated that mahanine can reduce the concentration of cisplatin to exhibit enhanced cytotoxicity in cervical cancer cells as reflected by regulation of several molecular mediators of apoptosis. The amplification of mitochondrial transmembrane depolarization, Bid truncation and activation of caspase-9 in combination-treated cells confirmed the synergistic-interactive effects of cisplatin and mahanine. It is known that the HeLa belong to the Type II of cancer cell lines. It could be the reason why cisplatin-induced apoptosis was more in HeLa than SiHa. Furthermore, caspase-9 cleavage was also seen primarily in the cisplatin-treated HeLa cells. Interestingly, in HeLa, Bid cleavage after combination-treatment in contrast to SiHa suggested a small difference in extrinsic pathway activation. This may be due to the more expression of basal level Fas in HeLa than SiHa cells (Fig. S-5) as anti-CD95 showed a little more binding with HeLa. Additionally, we blocked the surface FasL with FasLneutralizing antibody (NOK-2) and observed more inhibition of cell death in HeLa further suggesting the additional activation of extrinsic apoptotic machinery (Fig. S-4). The constitutive phosphorylation at Tyr705 of STAT3 mainly confers drug resistance to several cancers [28]. Activated STAT3 is also linked with cisplatin-resistance in head and neck squamous cell carcinoma [29]. These make STAT3 an effective target for cancer
therapy [26]. Here, we observed the ability of mahanine to deactivate STAT3 by reducing its Tyr705 phosphorylation and subsequent decreasing its nuclear translocation. Thus it could be hypothesised that mahanine may enhance chemo-sensitivity of cervical cancer cells to cisplatin by reducing STAT3 activity. In this perspective, we combined mahanine and cisplatin at fixed ratio and observed enhanced accumulation of cytosolic p-STAT3 and inhibition of nuclear translocation, although cisplatin alone had little effect on STAT3 deactivation under this condition. We already demonstrated that mahanine mediates ubiquitination of p-STAT3 leading to its reduction in SiHa cells. It might be possible that combination treatment resulted in ubiquitination of p-STAT3 in advance as compared to the individual treatments due to enhancement of their cytotoxic effect. This could be the reason that the robust enhancement of p-STAT3 does not occur after combined exposure in cytosolic portion of SiHa cells in comparison to single agent alone. Furthermore, cisplatin, at lower concentration, was unable to decrease expressions of cell proliferation-related proteins (c-myc, c-Jun). However, at the same dose, in combination with mahanine, cisplatin showed inhibition of all these proteins. It is reported that STAT3 downregulates tumour-suppressor pathway proteins in melanoma and colon cancer [30] and previously we reported mahanine-mediated activation of p53 in colon cancer [21]. Therefore, it may be envisaged a possible link between involvement of STAT3 inactivation and activation of p53 in mahanine-treated cancer cells. ROS generation is one of the key mechanisms of cisplatininduced apoptosis [31]. Mahanine-induced ROS [17] played an
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 8
R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
Fig. 5. Mahanine suppressed STAT3 activation and promoted ubiquitin-dependent proteasomal degradation of p-STAT3 Tyr 705. (A) Cells were exposed to increasing concentrations of mahanine (0–16 lM) for 24 h. After treatment cells were sonicated, cell lysates were electrophorsed and analysed by Western blot using anti-STAT3, pSTAT3 (Tyr705), Src, p-Src, JAK1, p-JAK1 antibodies. (B) Equal amount of proteins (40 lg) of cytosolic and nuclear fractions of mahanine (0–16 lM)-treated cells after 24 h were separated by SDS–PAGE and proteins were analysed by immunoblotting with anti-STAT3 and p-STAT3 antibodies. (C) For the detection of proteasome-mediated degradation, HeLa and SiHa cells were briefly exposed for 1 h in presence and absence to MG132 (10 lM), a proteasome inhibitor. Next they were kept for 24 h in presence and absence of mahanine (12 lM). Cell lysates were prepared, electrophorsed and immunoblotted with anti-p-STAT3 (Tyr705) and anti-STAT3 antibodies. (D) To identify pSTAT3 ubiquitination, MG132 (10 lM, 1 h) pretreated cells were further exposed to mahanine (12 lM) for 24 h. Cell lysates were immunoprecipitated with anti-p-STAT3 (Tyr705) antibody. The precipitated proteins were electrophorsed under reducing condition and immunoblotted with anti-ubiquitin (Ub) antibody. Each experiment was done at least three times.
Fig. 6. Mahanine in combination with cisplatin induced enhanced deactivation of STAT3 and its downstream proteins. Cells were exposed to either cisplatin (3 lM) or mahanine (12 lM) alone or their combination at 1:4 (cisplatin:mahanine) for 24 h. (A) Cell lysates of treated cells were electrophorsed and analysed by Western blot with anti-STAT3, p-STAT3 (Tyr705), Src, p-Src, JAK1, p-JAK1 antibodies. (B) Mahanine exposed cells were separated into cytosol and nuclear portions and equal amount of proteins (40 lg) of both fractions were analysed by Western blot with anti-p-STAT3 (Tyr705) and anti-STAT3 antibodies. (C) Cell lysates of treated cells were explored by Western blot analysis with anti-c-myc and c-Jun antibodies.
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
9
Fig. 7. Combination of mahanine and cisplatin induced enhanced inhibition of cervical cancer cell migration. (A) Cells were exposed to either cisplatin (3 lM) or mahanine (12 lM) alone or their combination at ratio of 1:4 (cisplatin:mahanine) for 24 h. Cell lysates was prepared, electrophorsed and immunoblotted with anti-MMP9 and antiVEGF antibodies. (B) HeLa and SiHa cells were seeded in 6-well plate and grown to >90% confluency. Confluent monolayer of both cells were scraped with micropipette tip and exposed to either cisplatin (3 lM) or mahanine (12 lM) alone or their combination for 24 h. Cells migrated to wounded region were assessed by phase contrast microscopy. The inhibition of migration was expressed by calculating the area of wound which was represented as Bar graph. Each value was the mean ± SD of independent experiments. ‘‘’’ indicated a significant difference of p < 0.05.
433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457
important role in Hsp90 inhibition in pancreatic cancer [18], PTEN activation in colon cancer [21] and G0–G1 phase arrest in glioblastoma multiforme [personal communication]. However, the role of ROS in mahanine-mediated STAT3 deactivation is yet to be investigated. Natural compounds are reported to suppress STAT3 activation either through direct binding to its SH2 domain or by inhibition of upstream tyrosine kinases JAK1 and Src and/or up regulation of SHP-1 and SHP-2 [32,33]. Mahanine-mediated suppression of JAK1 and Src might play a significant role in the inhibition of phosphorylation of STAT3. Inhibition of both Src and JAK exhibited similar down regulation of p-STAT3 level as mahanine in combination with cisplatin suggesting their role in this event under the experimental condition. Ubiquitin-dependent proteasomal degradation of STAT3 is involved as negative regulator of this pathway in hepatocellular carcinoma [27]. Here, we also observed that mahanine promotes ubiquitin-dependent proteasome-mediated degradation of p-STAT3. Therefore, our data convincingly demonstrated that mahanine induces apoptosis of cervical cancer cells through inhibition of tyrosine kinases followed by reduction of p-STAT3 leading to its proteasome-dependent degradation. Moreover, combination treatment resulted in enhanced suppression of upstream regulators and subsequent inhibition of p-STAT3. PTEN is reported to act as a negative controller of STAT3 activation [34]. Mahanine-mediated PTEN activation in colon cancer [21] suggested
that PTEN may also be involved in the inhibition of STAT3 in treated cervical cancer cells. Furthermore, down regulation of MMP9 after combinationtreatment confirmed the involvement of mahanine-mediated STAT3 deactivation and further verified its anti-metastatic activity [18]. This is corroborated by enhanced inhibition of cell migration after combination treatment. In conclusion, we demonstrated that nontoxic herbal compound; mahanine synergistically potentiates the growth-inhibitory activity of cisplatin in cervical cancer and significantly reduces its concentrations. The combination of mahanine and cisplatin induced enhanced inhibition of STAT3 activation, cell migration and increased apoptosis of cervical cancer cells compared to single agent. Taken together, our findings highlight another major application of mahanine in reducing the concentration of cisplatin for its wider use.
458
Conflict of Interest
474
The authors declare no conflicts of interest.
459 460 461 462 463 464 465 466 467 468 469 470 471 472 473
475
Acknowledgements
476
This work received financial support from CSIR-IICB, CSIR under IAP-0001, HCP004, NMITLI, TLP-004 and DBT (GAP 235), ICMR,
477
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
478
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 10
R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
481
Govt. of India. Dr. Chitra Mandal is grateful to financial support by Sir J.C. Bose Fellowship, DST of Govt. of India and also mutual grant from ICMR and German Cancer Research Centre.
482
Appendix A. Supplementary data
483 485
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canlet.2014.05. 005.
486
References
487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534
[1] International Agency for Research on Cancer, Lyon, France, 2010. . [2] D.R. Lowy, J.T. Schiller, Prophylactic human papillomavirus vaccines, J. Clin. Invest. 116 (2006) 1167–1173. [3] J.T. Schiller, X. Castellsagué, L.L. Villa, A. Hildesheim, An update of prophylactic human papillomavirus L1 virus-like particle vaccine clinical trial results, Vaccine 19 (2008) 26. [4] J.H. Maduro, E. Pras, P.H. Willemse, E.G. de Vries, Acute and long-term toxicity following radiotherapy alone or in combination with chemotherapy for locally advanced cervical cancer, Cancer Treat. Rev. 29 (2003) 471–488. [5] Z.H. Siddik, Cisplatin: mode of cytotoxic action and molecular basis of resistance, Oncogene 22 (2003) 7265–7279. [6] Y. Li, F. Ahmed, S. Ali, P.A. Philip, O. Kucuk, F.H. Sarkar, Inactivation of nuclear factor kappa B by soy isoflavone genistein contributes to increased apoptosis induced by chemotherapeutic agents in human cancer cells, Cancer Res. 65 (2005) 6934–6942. [7] F. He, Q. Wang, X.L. Zheng, J.Q. Yan, L. Yang, H. Sun, L.N. Hu, Y. Lin, X. Wang, Wogonin potentiates cisplatin-induced cancer cell apoptosis through accumulation of intracellular reactive oxygen species, Oncol Rep. 28 (2012) 601–605. [8] X. Wang, S. Govind, S.P. Sajankila, L. Mi, R. Roy, F.L. Chung, Phenethyl isothiocyanate sensitizes human cervical cancer cells to apoptosis induced by cisplatin, Mol. Nutr. Food Res. 55 (2011) 1572–1581. [9] H. Yu, R. Jove, The STATs of cancer-new molecular targets comes of age, Nat. Rev. Cancer 4 (2004) 97–105. [10] C.L. Chen, F.C. Hsieh, J.C. Lieblein, J. Brown, C. Chan, J.A. Wallace, G. Cheng, B.M. Hall, J. Lin, Stat3 activation in human endometrial and cervical cancers, Br. J. Cancer 96 (2007) 591–599. [11] S. Huang, Regulation of metastases by signal transducer and activator of transcription 3 signaling pathway: clinical implications, Clin. Cancer Res. 13 (2007) 1362–1366. [12] D.J. Stewart, Mechanisms of resistance to cisplatin and carboplatin, Crit. Rev. Oncol. Hematol. 63 (2007) 12–31. [13] X. Yao, F. Zhu, Z. Zhao, C. Liu, L. Luo, Z.J. Yin, Arctigenin enhances chemosensitivity of cancer cells to cisplatin through inhibition of the STAT3 signaling pathway, Cell Biochem. 112 (2011) 2837–2849. [14] S. Sinha, B.C. Pal, S. Jagadeesh, P.P. Banerjee, A. Bandyopadhaya, S. Bhattacharya, Mahanine inhibits growth and induces apoptosis in prostate cancer cells through the deactivation of Akt and activation of caspases, Prostate 66 (2006) 1257–1265. [15] C. Mandal, B.C. Pal, K. Bhattacharya, S.K. Samanta, S. Sarkar, R. Das. Process for the isolation of organic compounds useful for the treatment of cancer, US Patent (2013) Application Publication No.: US 2013/0065932 A1 Pub. Date: March 14. [16] M.K. Roy, V.N. Thalang, G. Trakoontivakorn, K. Nakahara, Mahanine, a carbazole alkaloid from Micromelum minutum, inhibits cell growth and induces apoptosis in U937 cells through a mitochondrial dependent pathway, Br. J. Pharmacol. 145 (2005) 145–155.
479 480
484
[17] K. Bhattacharya, S.K. Samanta, R. Tripathi, A. Mallick, S. Chandra, B.C. Pal, C. Shaha, C. Mandal, Apoptotic effects of mahanine on human leukemic cells are mediated through crosstalk between Apo-1/Fas signaling and the Bid protein and via mitochondrial pathways, Biochem. Pharmacol. 79 (2010) 361–372. [18] S. Sarkar, D. Dutta, S.K. Samanta, K. Bhattacharya, B.C. Pal, J. Li, K. Datta, C. Mandal, C. Mandal, Oxidative inhibition of Hsp90 disrupts the superchaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo, Int. J. Cancer 132 (2013) 695–706. [19] S.K. Samanta, D. Dutta, S. Roy, K. Bhattacharya, S. Sarkar, A.K. Dasgupta, B.C. Pal, C. Mandal, C. Mandal, Mahanine, a DNA minor groove binding agent exerts cellular cytotoxicity with involvement of C-7-OH and -NH functional groups, J. Med. Chem. 56 (2013) 5709–5721. [20] S. Jagadeesh, S. Sinha, B.C. Pal, S. Bhattacharya, P.P. Banerjee, Mahanine reverses an epigenetically silenced tumor suppressor gene RASSF1A in human prostate cancer cells, Biochem. Biophys. Res. Commun. 362 (2007) 212–217. [21] R. Das, K. Bhattacharya, S. Sarkar, S.K. Samanta, B.C. Pal, C. Mandal, Mahanine synergistically enhances cytotoxicity of 5-fluorouracil through ROS-mediated activation of PTEN and p53/p73 in colon carcinoma, Apoptosis 19 (2014) 149– 164. [22] T.C. Chou, Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies, Pharmacol. Rev. 58 (2006) 621–681. [23] C. Mandal, A. Dutta, A. Mallick, S. Chandra, L. Misra, R.S. Sangwan, C. Mandal, Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade, Apoptosis 13 (2008) 1450–1464. [24] G. Kroemer, L. Galluzzi, C. Brenner, Mitochondrial membrane permeabilization in cell death, Physiol. Rev. 87 (2007) 99–163. [25] S. Shukla, G. Shishodia, S. Mahata, S. Hedau, A. Pandey, S. Bhambhani, S. Batra, S.F. Basir, B.C. Das, A.C. Bharti, Aberrant expression and constitutive activation of STAT3 in cervical carcinogenesis: implications in high-risk human papillomavirus infection, Mol. Cancer 9 (2010) 282. [26] X. Wang, P.J. Crowe, D. Goldstein, J.L. Yang, STAT3 inhibition, a novel approach to enhancing targeted therapy in human cancers (review), Int. J. Oncol. 41 (2012) 1181–1191. [27] K. Selvendiran, H. Koga, T. Ueno, T. Yoshida, M. Maeyama, T. Torimura, H. Yano, M. Kojiro, M. Sata, Luteolin promotes degradation in signal transducer and activator of transcription 3 in human hepatoma cells: an implication for the antitumor potential of flavonoids, Cancer Res. 66 (2006) 4826–4834. [28] B. Barré, A. Vigneron, N. Perkins, I.B. Roninson, E. Gamelin, O. Coqueret, The STAT3 oncogene as a predictive marker of drug resistance, Trends Mol Med. 13 (2007) 4–11. [29] F. Gu, Y. Ma, Z. Zhang, J. Zhao, H. Kobayashi, L. Zhang, L. Fu, Expression of Stat3 and Notch1 is associated with cisplatin resistance in head and neck squamous cell carcinoma, Oncol. Rep. 23 (2010) 671–676. [30] G. Niu, K.L. Wright, Y. Ma, G.M. Wright, M. Huang, R. Irby, J. Briggs, J. Karras, W.D. Cress, D. Pardoll, R. Jove, J. Chen, H. Yu, Role of Stat3 in regulating p53 expression and function, Mol. Cell. Biol. 25 (2005) 7432–7440. [31] L. Wang, P. Chanvorachote, D. Toledo, C. Stehlik, R.R. Mercer, V. Castranova, Y. Rojanasakul, Peroxide is a key mediator of Bcl-2 down-regulation and apoptosis induction by cisplatin in human lung cancer cells, Mol. Pharmacol. 73 (2008) 119–127. [32] D.S. Shin, H.N. Kim, K.D. Shin, Y.J. Yoon, S.J. Kim, D.C. Han, B.M. Kwon, Cryptotanshinone inhibits constitutive signal transducer and activator of transcription 3 function through blocking the dimerization in DU145 prostate cancer cells, Cancer Res. 69 (2009) 193–202. [33] F. Li, P.P. Fernandez, P. Rajendran, K.M. Hui, G. Sethi, Diosgenin, a steroidal saponin, inhibits STAT3 signaling pathway leading to suppression of proliferation and chemosensitization of human hepatocellular carcinoma cells, Cancer Lett. 292 (2010) 197–207. [34] S. Sun, B.M. Steinberg, PTEN is a negative regulator of STAT3 activation in human papillomavirus-infected cells, J. Gen. Virol. 83 (2002) 1651–1658.
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597
No. of Pages 10, Model 5G
17 May 2014 Cancer Letters xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet 5 6
Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition
3 4 7 8 9
10 1 5 2 2 13 14 15 16 17 18 19 20 21 22 23 24
Q1
Ranjita Das a, Kaushik Bhattacharya a, Suman K. Samanta a, Bikas C. Pal b, Chitra Mandal a,⇑ a b
Cancer Biology and Inflammatory Disorder Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, Kolkata, India National Institute of Pharmaceutical Education and Research, Kolkata, India
a r t i c l e
i n f o
Article history: Received 8 January 2014 Received in revised form 11 April 2014 Accepted 1 May 2014 Available online xxxx Keywords: Mahanine Cisplatin Cervical cancer Synergism STAT3
a b s t r a c t Toxicity reduction of cisplatin is necessary for improved treatment of cancer. Here we have demonstrated the synergistic growth-inhibitory effect of cisplatin on cervical cancer cells in-combination with a nontoxic herbal carbazole alkaloid, mahanine. Mahanine enhanced cisplatin-induced apoptosis and reduced its effective concentration 5–8 folds. Mahanine inhibited JAK1 and Src and subsequently promoted proteasome-mediated degradation of STAT3. This event was further enhanced in-combination with cisplatin and subsequently inhibited cancer cell migration. Collectively, our results revealed that mahanine may be a prospective agent to reduce the concentration of cisplatin in adjunct for the treatment of cancer and thereby decreasing its toxicity. Ó 2014 Published by Elsevier Ireland Ltd.
26 27 28 29 30 31 32 33 34 35
36 37 38
Introduction
39
Cervical cancer is the third most common malignancy worldwide and it comprises of 13% of all cancers [1]. Human papilloma virus (HPV) infection with high risk type is the main factor for the development of cervical cancer. Among 15 subtypes of highrisk HPV strains, types 16 and 18 are mainly responsible for 76.7% of this cancer in Indian women [2]. Recently, two prophylactic HPV vaccines (Gardasil and Cervarix) are in the market. However, they have no benefit over HPV infection or cervical cancer [3]. Thus, new strategies are still needed. Current treatments are radiotherapy, chemotherapy and surgery. Cisplatin is the drug of choice either alone or in combination with topotecan. However, severe side-effects like bone-marrow depression, neutropenia, thrombocytopenia and anaemia due to haematological toxicity along with nephrotoxicity and neurotoxicity [4] and acquired chemoresistance [5] throughout the course of treatment have limited the usage of cisplatin. To conquer the restrictions in clinical use of cisplatin, combined therapy with dietary compounds is recommended [6–8]. Signal Transducer and Activator of Transcription 3 (STAT3) plays an important role in cellular survival, proliferation, and
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
⇑ Corresponding author. Address: Cancer Biology and Inflammatory Disorders Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India. Tel.: +91 33 2429 8861; fax: +91 33 2473 5197. E-mail addresses: [email protected], [email protected] (C. Mandal).
differentiation. Constitutively activated STAT3 is found in breast, prostrate, lung, ovarian, head and neck, leukemia, lymphoma and cervical cancers etc. [9–10]. Upstream activators of STAT3 include epidermal growth factor receptor (EGFR), IL-6 receptors or nonreceptor tyrosine kinases, Src or Janus kinase (JAK). Phosphorylation at tyrosine (Tyr) residue 705 activates STAT3 resulting in its dimerization, nuclear translocation, DNA binding and transcriptional activation of target genes. Activated STAT3 acts as a proto-oncogene by regulating proliferation and apoptosisassociated proteins such as cyclin D1, c-myc, survivin, Bcl-xL, Bcl-2 and Fas. STAT3 also increases invasion and metastasis by inducing matrix metalloproteinases (MMPs) and angiogenesis by the activation of VEGF and HIF-1a [11]. Overexpressed STAT3 confers resistance to cisplatin-induced apoptosis in cancers [12]. Therefore, inhibition of STAT3 activation may possibly sensitize cancer cells to cisplatin-induced apoptosis [13]. Mahanine, a carbazole alkaloid, purified from well-known Indian herbal plant Murraya koenigii, induced apoptosis in histiocytic lymphoma, promylocytic leukemia and prostate cancer cells [14,15]. In leukemic cells, mahanine induced apoptosis through mitochondrial death cascade and Fas-FasL activation [16,17]. In pancreatic cancer, it inhibited Hsp90 through reactive oxygen species (ROS) [18]. C-7-OH and 9-NH are two functional groups of mahanine. They were responsible for its cytotoxicity and minor groove binding with DNA [19]. It reduced in vivo xenograft [17] and orthotopic [18] tumours and showed nontoxicity to total body mass of normal Balb/c and athymic nude mice [15]. It activated
http://dx.doi.org/10.1016/j.canlet.2014.05.005 0304-3835/Ó 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 2 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106
R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
epigenetically suppressed tumour suppressor gene RASSF1A [20]. Recent study reported that mahanine and 5-fluorouracil combination exert synergistic increase in colon cancer cell death coupled with activation of tumour suppressor proteins, PTEN and p53/ p73 [21]. Hence, we wanted to explore this as a candidate for adjunct therapy with cisplatin in cervical cancer. Here, we have investigated that mahanine exerts anti-proliferative activity in cervical cancer cells alone. It potentially induced apoptosis in combination with cisplatin and reduced the concentration of this toxic drug by 5–8 folds thereby synergistically enhances its effect. The combination-induced apoptosis involved activation of caspase and mitochondria. Furthermore, mahanine potentially deactivated STAT3 by suppressing its phosphorylation. The decreased expression of JAK1 and Src might play important role in deactivation of STAT3. Mahanine also promoted ubiquitindependent proteasome-mediated p-STAT3 degradation. In combination with cisplatin, mahanine enhanced further inhibition of STAT3 phosphorylation, its upstream JAK1 and Src and cancer cell migration. Thus, mahanine could be a potent and valuable chemotherapeutic agent to reduce the toxicity of cisplatin by decreasing its definite concentration.
where (DX)1 and (DX)2 were the concentrations of each drug alone to exert x% effect and (D)1 and (D)2 were the drugs in combination to elicit the same effect. CI < 1, = 1 and > 1 indicate synergism, additivity and antagonism respectively. We further evaluated the fold decrease of each drug as a result of synergism represented as drugreduction index (DRI).
150 151 152 153 154
Apoptosis assay
155
Externalizations of phosphatidylserine were verified by double staining the cells with FITC-annexin V and PI [21]. Briefly, both cells (1 106) were treated either with cisplatin (3 lM) and mahanine (12–20 lM) alone or in combination for 24 h. Next, washed cells were resuspended in annexin-V binding buffer, kept for 45 min at 25 °C followed by incubation with FITC-annexin V and PI (5 lg/ml) for 20 min at 4 °C in dark. Cells were acquired and analysed with CellQuest Pro software on a FACSCalibur flow cytometer (BD).
156 157 158 159 160 161 162
Measurement of mitochondrial membrane potential
163
6
Both cells (1 10 ) were exposed to cisplatin (3 lM) and mahanine (12 lM) alone or their combination for 24 h, washed and incubated with JC1 (25 lM) for 30 min in dark at 37 °C. Cells were analysed by FACS to determine mitochondrial transmembrane depolarization [17] which was represented as mean fluorescence intensity (MFI) of green fluorescence.
164 165 166 167 168
Subcellular fractionation
169 6
107
Materials and methods
108
Reagents
109 110 111 112 113 114 115 116 117 118 119 120 121
The primary antibodies against phospho-STAT3 (Tyr705), STAT3, phosphoJAK1, JAK1, phopho-c-myc, c-myc, cyclin D1, MMP9, Bak, Bid, Bcl-xl, Fas, Caspase-8, Caspase-9, b-actin, poly-ADP ribose polymerase (PARP) and horseradish peroxidase (HRP)-linked secondary antibodies were from Cell Signalling Technology, USA. Primary antibody HDAC-3, FITC-annexin V and Mitoscreen kit (JC1) were from BD Bioscience (San Diego, USA). IMDM cell culture medium, FCS, antibiotic–antimycotic, and trypsin–EDTA were from Invitrogen, USA. Cisplatin, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI), molecular grade BSA, protein A-Sepharose 4B, Tween-20 and dimethyl sulphoxide (DMSO), Src Inhibitor-1 and cucurbitacin B hydrate (JAK inhibitor) were from Sigma–Aldrich, USA. NE-PER nuclear and cytoplasmic extraction reagents and SuperSignal West Pico imaging system were from Thermo-scientific, USA.
122
Mahanine purification and its characterization
123 124 125 126
Mahanine was purified as described earlier [14] from fresh leaves of an Indian plant, M. koenigii (Rutaceae family). The purity was confirmed by HPLC and LC-MS. 1 [H] and 13[C] NMR spectral data analyses established its structure as mahanine (Fig. 1A).
127
Cell lines and cell culture
128 129 130 131
Cervical cancer cell lines, HeLa (HPV-18) and SiHa (HPV-16) were obtained from American Type Culture Collection (Manassas, VA). The cells were grown in IMDM supplemented with 10% FCS and 1% antibiotic antimycotic mixture in a humidified incubator at 37 °C containing 5% CO2.
132
Cytotoxicity assay
133 134 135 136 137 138 139
The cytotoxicity of mahanine and cisplatin were assessed by MTT assay as described previously [18]. Briefly, HeLa and SiHa (1 104/well) cells were treated with mahanine (0–24 lM) and cisplatin (0–20 lM) for 24–48 h. MTT (0.4 mg/ml) was added and further incubated for 3 h in dark at 37 °C. The generated formazan complexes were dissolved in DMSO and the absorbance was measured at 550 nm with spectrophotometer (Thermo). Cell viability was derived from percentage of MTT conversion in treated relative to untreated cells defined as 100%.
140
Combination study with cisplatin
141 142 143 144 145 146
HeLa and SiHa (1 104) cells were treated with cisplatin (0–6 lM) and mahanine (2–24 lM) alone or in combination at a fixed concentration ratio of 1:4 (cisplatin:mahanine). The inhibitory effect was assessed after 48 h by MTT assay and evaluated based on combination index (CI). It was calculated using Chou–Talalay equation by Calcusyn software version 2.1 (Biosoft) [21,22] using the following equation:
147 149
CI ¼ ðDÞ1 =ðDX Þ1 þ ðDÞ2 =ðDX Þ2
Both cells (1 10 ) were treated with cisplatin (3 lM) and mahanine (0–16 lM) or with their combination for 24 h. Treated cells were separated into cytosol and nuclear fractions by NE-PERÒ kit following manufacturer’s protocol. Briefly, washed cells were resuspended in cytosol extraction reagent, vortexed, centrifuged and the supernatant was collected as the cytosolic fraction. The pellet was incubated with nuclear extraction reagent, centrifuged and supernatant used as the nuclear fraction.
170 171 172 173 174 175 176
Western blot analysis
177
Both cells (1 106) were exposed to cisplatin (3 lM) and mahanine (0–16 lM) or combination for 24 h. The cells were washed, resuspended in PBS, sonicated (2 watt and 3 pulses) and followed by cold centrifugation at 10,000 g [23]. The supernatants were used as cell lysates for further experiments. Equal amounts of protein (50 lg) were electrophorsed on SDS–PAGE (7.5–12%) and electrotransferred onto nitrocellulose membranes. The membranes were blocked with TBS-2% BSA and probed with appropriate primary antibodies at 4 °C for overnight. The blots were washed with TBS containing 0.1% Tween-20 and processed using HRP-secondary antibodies. Signals were detected with the West-pico ECL system.
178 179 180 181 182 183 184 185 186
Immunoprecipitation
187
For the detection of proteasome-dependent ubiquitination of p-STAT3 (Tyr 705), SiHa cells (1 106) were pretreated with MG132 (10 lM) for 1 h followed by mahanine treatment (12 lM) for 24 h. Total cell lysates were prepared. Proteins (300 lg) were incubated with anti-p-STAT3 antibody (1:100) for overnight at 4 °C and further with Protein A-Sepharose 4B. The immune complex was resolved by SDS–PAGE (7.5%) and detected using anti-ubiquitin antibody.
188 189 190 191 192 193
Scratch-Wound assay
194
Both cells (1 106) were grown to >90% confluency and starved in reducedserum (0.5%) media for overnight. Next, three separate linear scratches were made by a 200 ll micropipette tip. Wounded monolayers were washed thrice to remove cell debris, kept in 10% FBS medium and incubated with cisplatin (3 lM) and mahanine (12 lM) alone or their combination for 24 h. Images were taken using phasecontrast microscopy.
195 196 197 198 199 200
Statistical analysis
201
The data were obtained from at least three independent experiments. All statistical analyses were performed by Microsoft Excel and GraphPad Prism and evaluated by paired t test. The results were represented as mean ±SD from independent experiments. P values 1 indicates antagonism. The CI values < 1 indicated that interaction between cisplatin (2–5 lM) and mahanine (8–20 lM) was synergistic in both HeLa and SiHa cells.
257 258 259 260 261 262 263 264 265 266 267 268 269
also showed more PARP and Bid cleavage compared to mahanine while cisplatin could not induce these cleavages (Fig. 3B). Involvement of mitochondria is reported in mahanine-induced apoptosis which is a prooxidant molecule [17] and moreover ROS accumulation in mitochondrial lumen leads to the alteration of mitochondrial inner membrane potential [24]. This prompted us to measure mitochondrial membrane potential. Combination resulted in enhanced membrane depolarization signified by the higher MFI of green fluorescence in both cells (Fig. 4). Taken together, these suggested that the combination induces potential apoptosis in cervical cancer cells. Moreover, caspase activation and loss of mitochondrial transmembrane potential attributed to this apoptosis.
270
Mahanine suppressed STAT3 activation
271
Constitutive activation of STAT3 signalling is reported in cervical carcinoma [25] and inhibition of this may play a role in cancer interference [26]. In this context, we investigated whether mahanine can inhibit the STAT3 activation in these cells. Mahanine significantly inhibited phosphorylation of STAT3 (Tyr705) in a concentration-dependent manner. Besides, the total protein levels of STAT3 were almost unchanged, however, decreased only at 16 lM of mahanine (Fig. 5A). Src and JAK tyrosine kinases contribute in the activation of STAT3 [11]. To further elucidate mahanine-mediated suppression of p-STAT3, expression of total and phosphorylated Src and JAK1 proteins were checked. Mahanine resulted in a reduction of phosphorylation of Src and JAK1 in a concentration-dependent manner. The total Src expression was also decreased after mahanine treatment. Though, no significant inhibition was observed in total JAK1 (Fig. 5A). Tyr705 phosphorylation activates STAT3, promotes its dimerization and nuclear localization [9]. We found that mahanine
272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288
reduces the levels of total and phosphorylated STAT3 in nucleus whereas it was increased in cytosol (Fig. 5B). Therefore, we suggested that mahanine induces inhibition of STAT3 signalling in cervical cancer.
289
Mahanine promoted ubiquitin-dependent proteasome-mediated degradation of p-STAT3 (Tyr705)
293
Turnover of p-STAT3 is reported to be regulated by the ubiquitin-dependent proteasomal degradation [27]. Therefore, next we investigated any involvement of ubiquitin-dependent degradation in p-STAT3. MG132 pre-treated HeLa and SiHa cells restored both phosphorylated and total STAT3 (Fig. 5C). Furthermore, enhanced polyubiquitination of p-STAT3 was observed in the presence of MG132 in SiHa cells (Fig. 5D). These findings suggested that mahanine mediates ubiquitination of p-STAT3 leading to its reduction in cervical cancer cells.
295
Cisplatin in-combination with mahanine enhanced deactivation of STAT3 and its downstream proteins
304
Over expression of STAT3 is reported to confer resistance in cisplatin-induced apoptosis [28]. By now, we demonstrated that mahanine suppresses STAT3 activation. Therefore, here we wanted to check combination-induced STAT3 regulation in this cancer. Total STAT3 and p-STAT3 status in both cells remained almost unaffected after cisplatin (3 lM) treatment (Fig. 6A). Similarly, mahanine (12 lM) alone also had little effect on total STAT3 and p-STAT3 in HeLa whereas reduction was observed in SiHa cells. However, combination-exposure considerably decreased phosphorylation of STAT3 in both cells (Fig. 6A). Additionally, cisplatin alone was unable to reduce total and phosphorylation of Src and JAK1 whereas mahanine could suppress these proteins. More importantly, combination-treatment enhanced the reduction of p-Src and p-JAK1. Similar inhibition was observed in presence of their respective inhibitors (Fig. S-6 A and B). We also checked the p-STAT3 level in cisplatin-treated cells along with the cells treated with Src and JAK inhibitors separately and found that both inhibitors inhibits p-STAT3 level whereas cisplatin at 3 lM concentration was unable to reduce the level of p-STAT3. Moreover, combination treatment blocked nuclear translocation of p-STAT3 more than when used them individually, while in cytosol this treatment resulted in the accumulation of p-STAT3 (Fig. 6B). STAT3 activation promotes tumorigenesis by regulating the expression of various gene products involved in cell survival, proliferation and angiogenesis [11]. Accordingly, we wanted to ensure the status of a few downstream proteins of STAT3 after combination treatment. The combination-exposure enhanced the decrease of total and phosphorylated c-myc and c-Jun both in HeLa and SiHa cells than single agent. In contrast, c-myc and c-Jun were almost unchanged in single agent treatment (Figs. 6C and S-3). Moreover, among the angiogenic factors regulated by STAT3, we checked VEGF expression which was also decreased significantly after combination treatment (Fig. S-2). Altogether these results suggested
306
Table 3 Dose reduction index (DRI) values for cisplatin in combination with mahanine. Cell lines
Drug:compound
Molar ratio (cisplatin:mahanine)
DRI (75% fraction affected level)
HeLa SiHa
Cisplatin:mahanine Cisplatin:mahanine
1:4 1:4
4.991 8.369
The DRI signifies the fold decrease of cisplatin as a result of synergistic combination in compared with the concentration of a single agent needed to achieve the same effect. Concentrations of cisplatin were reduced 5-folds in HeLa and 8-folds in SiHa to achieve a 75% inhibition of cell proliferation respectively when the cells were exposed to combination treatment at fixed molar ratios after 48 h.
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
290 291 292
294
296 297 298 299 300 301 302 303
305
307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 6
R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
Fig. 3. Mahanine and cisplatin combination induced more apoptosis in cervical cancer cells. (A) HeLa and SiHa cells were exposed to either cisplatin (3 lM) or mahanine (12 lM) alone or in combination at ratio of 1:4 (cisplatin:mahanine) for 24 h. Increased apoptosis by the combination treatment compared to single agent alone was assessed by FITC-annexin-V and PI positivity through flow cytometry. (B) Both cell lines were treated with either cisplatin (4 lM) or mahanine (16 lM) alone or their combination at 1:4 (cisplatin:mahanine) for 24 h. Treated cells were sonicated to prepare cell lysates, electrophorsed and analysed by Western blot with anti-caspase 8, 9, Bid, PARP antibodies as described in Materials and Methods. Each experiment was done at least three times.
340 341
enhancement of STAT3 deactivation in combination-treated cervical cancer cells.
342
Combination of mahanine and cisplatin decreased more cell motility
343
352
MMPs play a pivotal role in cancer cell motility and invasion among which, MMP-9 is the transcriptional targets of STAT3 [11]. In this perspective, we examined the status of MMP-9 and demonstrated more decreased expression of it in the combination-treated cells (Fig. 7A). Furthermore, scratch-wound assay showed additional decreased motility of both cells after combination-treatment as indicated by unchanged area of wound. Combination treated cells were migrated little compared to single agent (Fig. 7B). These data assured that cervical cancer cell motility is inhibited more when cisplatin was used in combination with mahanine.
353
Discussion
354
The main objective of our study was to reduce the concentration of known anticancer drug (cisplatin) widely used for treat-
344 345 346 347 348 349 350 351
355
ment of different cancers. Accordingly, we have used a nontoxic herbal carbazole alkaloid, mahanine along with cisplatin. Here, we have used cervical cancer as a model system. The main achievement of our study is to establish mahanine as a synergistic modulator of cisplatin in cervical cancer. Mahanine and cisplatin alone exerted growth-inhibitory activity against these cancer cells. The importance of this study is that it synergistically augmented the cytotoxicity of cisplatin even at 5–8-fold reduced concentration. Mahanine alone inhibited STAT3 activation whereas; cisplatin under identical condition had little effect on STAT3 deactivation. However, mahanine in-combination with cisplatin, at the same dose, enhanced inhibition of STAT3 activation, augmented proteasome-mediated degradation and reduced migration of cervical cancer cells compared to single agent treatment. Our study gives us a new hope to reduce the concentration of a well known anti-cancer drug (cisplatin). Severe toxicity and acquired drug resistance are associated with cisplatin treatment [5]. Combination of such toxic chemotherapeutics with nontoxic naturally occurring compounds having anticancer properties would be new strategy to overcome this problem [21]. We demonstrated earlier that mahanine exhibits
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
7
Fig. 4. Mahanine and cisplatin combination induced additional depolarization of mitochondrial membrane potential in cervical cancer. Cells were exposed to either cisplatin (3 lM) or mahanine (12 lM) alone or their combination at ratio of 1:4 (cisplatin:mahanine). After incubation, cells were washed stained with JC-1 and analysed by flow cytometry. Mitochondrial transmembrane depolarization was measured by the increased MFI of green fluorescence in combination treatment than in single agent alone.
377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404
anti-proliferative activity against several cancers both in vitro and in vivo [15–18]. More importantly, it was nontoxic towards normal tissues and total body mass of treated mice [15]. It also synergistically reduced the concentration of 5-fluorouracil for apoptosis of colon cancer cells [21]. Here, we demonstrated that mahanine can reduce the concentration of cisplatin to exhibit enhanced cytotoxicity in cervical cancer cells as reflected by regulation of several molecular mediators of apoptosis. The amplification of mitochondrial transmembrane depolarization, Bid truncation and activation of caspase-9 in combination-treated cells confirmed the synergistic-interactive effects of cisplatin and mahanine. It is known that the HeLa belong to the Type II of cancer cell lines. It could be the reason why cisplatin-induced apoptosis was more in HeLa than SiHa. Furthermore, caspase-9 cleavage was also seen primarily in the cisplatin-treated HeLa cells. Interestingly, in HeLa, Bid cleavage after combination-treatment in contrast to SiHa suggested a small difference in extrinsic pathway activation. This may be due to the more expression of basal level Fas in HeLa than SiHa cells (Fig. S-5) as anti-CD95 showed a little more binding with HeLa. Additionally, we blocked the surface FasL with FasLneutralizing antibody (NOK-2) and observed more inhibition of cell death in HeLa further suggesting the additional activation of extrinsic apoptotic machinery (Fig. S-4). The constitutive phosphorylation at Tyr705 of STAT3 mainly confers drug resistance to several cancers [28]. Activated STAT3 is also linked with cisplatin-resistance in head and neck squamous cell carcinoma [29]. These make STAT3 an effective target for cancer
therapy [26]. Here, we observed the ability of mahanine to deactivate STAT3 by reducing its Tyr705 phosphorylation and subsequent decreasing its nuclear translocation. Thus it could be hypothesised that mahanine may enhance chemo-sensitivity of cervical cancer cells to cisplatin by reducing STAT3 activity. In this perspective, we combined mahanine and cisplatin at fixed ratio and observed enhanced accumulation of cytosolic p-STAT3 and inhibition of nuclear translocation, although cisplatin alone had little effect on STAT3 deactivation under this condition. We already demonstrated that mahanine mediates ubiquitination of p-STAT3 leading to its reduction in SiHa cells. It might be possible that combination treatment resulted in ubiquitination of p-STAT3 in advance as compared to the individual treatments due to enhancement of their cytotoxic effect. This could be the reason that the robust enhancement of p-STAT3 does not occur after combined exposure in cytosolic portion of SiHa cells in comparison to single agent alone. Furthermore, cisplatin, at lower concentration, was unable to decrease expressions of cell proliferation-related proteins (c-myc, c-Jun). However, at the same dose, in combination with mahanine, cisplatin showed inhibition of all these proteins. It is reported that STAT3 downregulates tumour-suppressor pathway proteins in melanoma and colon cancer [30] and previously we reported mahanine-mediated activation of p53 in colon cancer [21]. Therefore, it may be envisaged a possible link between involvement of STAT3 inactivation and activation of p53 in mahanine-treated cancer cells. ROS generation is one of the key mechanisms of cisplatininduced apoptosis [31]. Mahanine-induced ROS [17] played an
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 8
R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
Fig. 5. Mahanine suppressed STAT3 activation and promoted ubiquitin-dependent proteasomal degradation of p-STAT3 Tyr 705. (A) Cells were exposed to increasing concentrations of mahanine (0–16 lM) for 24 h. After treatment cells were sonicated, cell lysates were electrophorsed and analysed by Western blot using anti-STAT3, pSTAT3 (Tyr705), Src, p-Src, JAK1, p-JAK1 antibodies. (B) Equal amount of proteins (40 lg) of cytosolic and nuclear fractions of mahanine (0–16 lM)-treated cells after 24 h were separated by SDS–PAGE and proteins were analysed by immunoblotting with anti-STAT3 and p-STAT3 antibodies. (C) For the detection of proteasome-mediated degradation, HeLa and SiHa cells were briefly exposed for 1 h in presence and absence to MG132 (10 lM), a proteasome inhibitor. Next they were kept for 24 h in presence and absence of mahanine (12 lM). Cell lysates were prepared, electrophorsed and immunoblotted with anti-p-STAT3 (Tyr705) and anti-STAT3 antibodies. (D) To identify pSTAT3 ubiquitination, MG132 (10 lM, 1 h) pretreated cells were further exposed to mahanine (12 lM) for 24 h. Cell lysates were immunoprecipitated with anti-p-STAT3 (Tyr705) antibody. The precipitated proteins were electrophorsed under reducing condition and immunoblotted with anti-ubiquitin (Ub) antibody. Each experiment was done at least three times.
Fig. 6. Mahanine in combination with cisplatin induced enhanced deactivation of STAT3 and its downstream proteins. Cells were exposed to either cisplatin (3 lM) or mahanine (12 lM) alone or their combination at 1:4 (cisplatin:mahanine) for 24 h. (A) Cell lysates of treated cells were electrophorsed and analysed by Western blot with anti-STAT3, p-STAT3 (Tyr705), Src, p-Src, JAK1, p-JAK1 antibodies. (B) Mahanine exposed cells were separated into cytosol and nuclear portions and equal amount of proteins (40 lg) of both fractions were analysed by Western blot with anti-p-STAT3 (Tyr705) and anti-STAT3 antibodies. (C) Cell lysates of treated cells were explored by Western blot analysis with anti-c-myc and c-Jun antibodies.
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
9
Fig. 7. Combination of mahanine and cisplatin induced enhanced inhibition of cervical cancer cell migration. (A) Cells were exposed to either cisplatin (3 lM) or mahanine (12 lM) alone or their combination at ratio of 1:4 (cisplatin:mahanine) for 24 h. Cell lysates was prepared, electrophorsed and immunoblotted with anti-MMP9 and antiVEGF antibodies. (B) HeLa and SiHa cells were seeded in 6-well plate and grown to >90% confluency. Confluent monolayer of both cells were scraped with micropipette tip and exposed to either cisplatin (3 lM) or mahanine (12 lM) alone or their combination for 24 h. Cells migrated to wounded region were assessed by phase contrast microscopy. The inhibition of migration was expressed by calculating the area of wound which was represented as Bar graph. Each value was the mean ± SD of independent experiments. ‘‘’’ indicated a significant difference of p < 0.05.
433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457
important role in Hsp90 inhibition in pancreatic cancer [18], PTEN activation in colon cancer [21] and G0–G1 phase arrest in glioblastoma multiforme [personal communication]. However, the role of ROS in mahanine-mediated STAT3 deactivation is yet to be investigated. Natural compounds are reported to suppress STAT3 activation either through direct binding to its SH2 domain or by inhibition of upstream tyrosine kinases JAK1 and Src and/or up regulation of SHP-1 and SHP-2 [32,33]. Mahanine-mediated suppression of JAK1 and Src might play a significant role in the inhibition of phosphorylation of STAT3. Inhibition of both Src and JAK exhibited similar down regulation of p-STAT3 level as mahanine in combination with cisplatin suggesting their role in this event under the experimental condition. Ubiquitin-dependent proteasomal degradation of STAT3 is involved as negative regulator of this pathway in hepatocellular carcinoma [27]. Here, we also observed that mahanine promotes ubiquitin-dependent proteasome-mediated degradation of p-STAT3. Therefore, our data convincingly demonstrated that mahanine induces apoptosis of cervical cancer cells through inhibition of tyrosine kinases followed by reduction of p-STAT3 leading to its proteasome-dependent degradation. Moreover, combination treatment resulted in enhanced suppression of upstream regulators and subsequent inhibition of p-STAT3. PTEN is reported to act as a negative controller of STAT3 activation [34]. Mahanine-mediated PTEN activation in colon cancer [21] suggested
that PTEN may also be involved in the inhibition of STAT3 in treated cervical cancer cells. Furthermore, down regulation of MMP9 after combinationtreatment confirmed the involvement of mahanine-mediated STAT3 deactivation and further verified its anti-metastatic activity [18]. This is corroborated by enhanced inhibition of cell migration after combination treatment. In conclusion, we demonstrated that nontoxic herbal compound; mahanine synergistically potentiates the growth-inhibitory activity of cisplatin in cervical cancer and significantly reduces its concentrations. The combination of mahanine and cisplatin induced enhanced inhibition of STAT3 activation, cell migration and increased apoptosis of cervical cancer cells compared to single agent. Taken together, our findings highlight another major application of mahanine in reducing the concentration of cisplatin for its wider use.
458
Conflict of Interest
474
The authors declare no conflicts of interest.
459 460 461 462 463 464 465 466 467 468 469 470 471 472 473
475
Acknowledgements
476
This work received financial support from CSIR-IICB, CSIR under IAP-0001, HCP004, NMITLI, TLP-004 and DBT (GAP 235), ICMR,
477
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
478
CAN 11885
No. of Pages 10, Model 5G
17 May 2014 10
R. Das et al. / Cancer Letters xxx (2014) xxx–xxx
481
Govt. of India. Dr. Chitra Mandal is grateful to financial support by Sir J.C. Bose Fellowship, DST of Govt. of India and also mutual grant from ICMR and German Cancer Research Centre.
482
Appendix A. Supplementary data
483 485
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canlet.2014.05. 005.
486
References
487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534
[1] International Agency for Research on Cancer, Lyon, France, 2010. . [2] D.R. Lowy, J.T. Schiller, Prophylactic human papillomavirus vaccines, J. Clin. Invest. 116 (2006) 1167–1173. [3] J.T. Schiller, X. Castellsagué, L.L. Villa, A. Hildesheim, An update of prophylactic human papillomavirus L1 virus-like particle vaccine clinical trial results, Vaccine 19 (2008) 26. [4] J.H. Maduro, E. Pras, P.H. Willemse, E.G. de Vries, Acute and long-term toxicity following radiotherapy alone or in combination with chemotherapy for locally advanced cervical cancer, Cancer Treat. Rev. 29 (2003) 471–488. [5] Z.H. Siddik, Cisplatin: mode of cytotoxic action and molecular basis of resistance, Oncogene 22 (2003) 7265–7279. [6] Y. Li, F. Ahmed, S. Ali, P.A. Philip, O. Kucuk, F.H. Sarkar, Inactivation of nuclear factor kappa B by soy isoflavone genistein contributes to increased apoptosis induced by chemotherapeutic agents in human cancer cells, Cancer Res. 65 (2005) 6934–6942. [7] F. He, Q. Wang, X.L. Zheng, J.Q. Yan, L. Yang, H. Sun, L.N. Hu, Y. Lin, X. Wang, Wogonin potentiates cisplatin-induced cancer cell apoptosis through accumulation of intracellular reactive oxygen species, Oncol Rep. 28 (2012) 601–605. [8] X. Wang, S. Govind, S.P. Sajankila, L. Mi, R. Roy, F.L. Chung, Phenethyl isothiocyanate sensitizes human cervical cancer cells to apoptosis induced by cisplatin, Mol. Nutr. Food Res. 55 (2011) 1572–1581. [9] H. Yu, R. Jove, The STATs of cancer-new molecular targets comes of age, Nat. Rev. Cancer 4 (2004) 97–105. [10] C.L. Chen, F.C. Hsieh, J.C. Lieblein, J. Brown, C. Chan, J.A. Wallace, G. Cheng, B.M. Hall, J. Lin, Stat3 activation in human endometrial and cervical cancers, Br. J. Cancer 96 (2007) 591–599. [11] S. Huang, Regulation of metastases by signal transducer and activator of transcription 3 signaling pathway: clinical implications, Clin. Cancer Res. 13 (2007) 1362–1366. [12] D.J. Stewart, Mechanisms of resistance to cisplatin and carboplatin, Crit. Rev. Oncol. Hematol. 63 (2007) 12–31. [13] X. Yao, F. Zhu, Z. Zhao, C. Liu, L. Luo, Z.J. Yin, Arctigenin enhances chemosensitivity of cancer cells to cisplatin through inhibition of the STAT3 signaling pathway, Cell Biochem. 112 (2011) 2837–2849. [14] S. Sinha, B.C. Pal, S. Jagadeesh, P.P. Banerjee, A. Bandyopadhaya, S. Bhattacharya, Mahanine inhibits growth and induces apoptosis in prostate cancer cells through the deactivation of Akt and activation of caspases, Prostate 66 (2006) 1257–1265. [15] C. Mandal, B.C. Pal, K. Bhattacharya, S.K. Samanta, S. Sarkar, R. Das. Process for the isolation of organic compounds useful for the treatment of cancer, US Patent (2013) Application Publication No.: US 2013/0065932 A1 Pub. Date: March 14. [16] M.K. Roy, V.N. Thalang, G. Trakoontivakorn, K. Nakahara, Mahanine, a carbazole alkaloid from Micromelum minutum, inhibits cell growth and induces apoptosis in U937 cells through a mitochondrial dependent pathway, Br. J. Pharmacol. 145 (2005) 145–155.
479 480
484
[17] K. Bhattacharya, S.K. Samanta, R. Tripathi, A. Mallick, S. Chandra, B.C. Pal, C. Shaha, C. Mandal, Apoptotic effects of mahanine on human leukemic cells are mediated through crosstalk between Apo-1/Fas signaling and the Bid protein and via mitochondrial pathways, Biochem. Pharmacol. 79 (2010) 361–372. [18] S. Sarkar, D. Dutta, S.K. Samanta, K. Bhattacharya, B.C. Pal, J. Li, K. Datta, C. Mandal, C. Mandal, Oxidative inhibition of Hsp90 disrupts the superchaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo, Int. J. Cancer 132 (2013) 695–706. [19] S.K. Samanta, D. Dutta, S. Roy, K. Bhattacharya, S. Sarkar, A.K. Dasgupta, B.C. Pal, C. Mandal, C. Mandal, Mahanine, a DNA minor groove binding agent exerts cellular cytotoxicity with involvement of C-7-OH and -NH functional groups, J. Med. Chem. 56 (2013) 5709–5721. [20] S. Jagadeesh, S. Sinha, B.C. Pal, S. Bhattacharya, P.P. Banerjee, Mahanine reverses an epigenetically silenced tumor suppressor gene RASSF1A in human prostate cancer cells, Biochem. Biophys. Res. Commun. 362 (2007) 212–217. [21] R. Das, K. Bhattacharya, S. Sarkar, S.K. Samanta, B.C. Pal, C. Mandal, Mahanine synergistically enhances cytotoxicity of 5-fluorouracil through ROS-mediated activation of PTEN and p53/p73 in colon carcinoma, Apoptosis 19 (2014) 149– 164. [22] T.C. Chou, Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies, Pharmacol. Rev. 58 (2006) 621–681. [23] C. Mandal, A. Dutta, A. Mallick, S. Chandra, L. Misra, R.S. Sangwan, C. Mandal, Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade, Apoptosis 13 (2008) 1450–1464. [24] G. Kroemer, L. Galluzzi, C. Brenner, Mitochondrial membrane permeabilization in cell death, Physiol. Rev. 87 (2007) 99–163. [25] S. Shukla, G. Shishodia, S. Mahata, S. Hedau, A. Pandey, S. Bhambhani, S. Batra, S.F. Basir, B.C. Das, A.C. Bharti, Aberrant expression and constitutive activation of STAT3 in cervical carcinogenesis: implications in high-risk human papillomavirus infection, Mol. Cancer 9 (2010) 282. [26] X. Wang, P.J. Crowe, D. Goldstein, J.L. Yang, STAT3 inhibition, a novel approach to enhancing targeted therapy in human cancers (review), Int. J. Oncol. 41 (2012) 1181–1191. [27] K. Selvendiran, H. Koga, T. Ueno, T. Yoshida, M. Maeyama, T. Torimura, H. Yano, M. Kojiro, M. Sata, Luteolin promotes degradation in signal transducer and activator of transcription 3 in human hepatoma cells: an implication for the antitumor potential of flavonoids, Cancer Res. 66 (2006) 4826–4834. [28] B. Barré, A. Vigneron, N. Perkins, I.B. Roninson, E. Gamelin, O. Coqueret, The STAT3 oncogene as a predictive marker of drug resistance, Trends Mol Med. 13 (2007) 4–11. [29] F. Gu, Y. Ma, Z. Zhang, J. Zhao, H. Kobayashi, L. Zhang, L. Fu, Expression of Stat3 and Notch1 is associated with cisplatin resistance in head and neck squamous cell carcinoma, Oncol. Rep. 23 (2010) 671–676. [30] G. Niu, K.L. Wright, Y. Ma, G.M. Wright, M. Huang, R. Irby, J. Briggs, J. Karras, W.D. Cress, D. Pardoll, R. Jove, J. Chen, H. Yu, Role of Stat3 in regulating p53 expression and function, Mol. Cell. Biol. 25 (2005) 7432–7440. [31] L. Wang, P. Chanvorachote, D. Toledo, C. Stehlik, R.R. Mercer, V. Castranova, Y. Rojanasakul, Peroxide is a key mediator of Bcl-2 down-regulation and apoptosis induction by cisplatin in human lung cancer cells, Mol. Pharmacol. 73 (2008) 119–127. [32] D.S. Shin, H.N. Kim, K.D. Shin, Y.J. Yoon, S.J. Kim, D.C. Han, B.M. Kwon, Cryptotanshinone inhibits constitutive signal transducer and activator of transcription 3 function through blocking the dimerization in DU145 prostate cancer cells, Cancer Res. 69 (2009) 193–202. [33] F. Li, P.P. Fernandez, P. Rajendran, K.M. Hui, G. Sethi, Diosgenin, a steroidal saponin, inhibits STAT3 signaling pathway leading to suppression of proliferation and chemosensitization of human hepatocellular carcinoma cells, Cancer Lett. 292 (2010) 197–207. [34] S. Sun, B.M. Steinberg, PTEN is a negative regulator of STAT3 activation in human papillomavirus-infected cells, J. Gen. Virol. 83 (2002) 1651–1658.
Please cite this article in press as: R. Das et al., Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.005
535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597
