Abstract Head and neck squamous cell carcinoma (HNSCC) remains difficult to treat, and despite of advances in treatment, the overall survival rate has only modestly improved over the past several years. Thus, there is an urgent need for additional therapeutic modalities. We hypothesized that treatment of HNSCC cells with a dietary product such as bitter melon extract (BME) modulates multiple signaling pathways and regresses HNSCC tumor growth in a preclinical model. We observed a reduced cell proliferation in HNSCC cell lines. The mechanistic studies reveal that treatment of BME in HNSCC cells inhibited c-Met signaling pathway. We also observed that BME treatment in HNSCC reduced phosphoStat3, c-myc and Mcl-1 expression, downstream signaling molecules of c-Met. Furthermore, BME treatment in HNSCC cells modulated the expression of key cell cycle progression molecules leading to halted cell growth. Finally, BME feeding in mice bearing HNSCC xenograft tumor resulted in an inhibition of tumor growth and c-Met expression. Together, our results suggested that BME treatment in HNSCC cells modulates multiple signaling pathways and may have therapeutic potential for treating HNSCC.

Citation: Rajamoorthi A, Shrivastava S, Steele R, Nerurkar P, Gonzalez JG, Crawford S, et al. (2013) Bitter Melon Reduces Head and Neck Squamous Cell Carcinoma Growth by Targeting c-Met Signaling. PLoS ONE 8(10): e78006. https://doi.org/10.1371/journal.pone.0078006 Editor: Sujit Basu, Ohio State University, United States of America Received: August 29, 2013; Accepted: September 16, 2013; Published: October 17, 2013 Copyright: © 2013 Rajamoorthi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grant R21CA137424 from the NIH, The Lottie Caroline Hardy Charitable Trust Fund, and Saint Louis University Cancer Center seed grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer in the world. Overall survival rate has not significantly improved in the past couple of decades, despite significant improvements in surgical procedures, radiotherapy, and chemotherapy [1]. In the United States, 50,000 new cases are diagnosed, and nearly 10,000 deaths are attributable to this disease annually [1]. HNSCCs are highly heterogeneous and contain a large number of genetic alterations which make them refractory to specific targeted drugs. The epidermal growth factor receptor (EGFR) is overexpressed in ∼90% of the HNSCC, and involved in cell growth, invasion, angiogenesis and metastasis [1], [2]. The c-Met pathway is also aberrantly upregulated in HNSCC, and activates the same downstream signaling pathway as EGFR. The ubiquitous expression of tyrosine kinase, such as EGFR and/or c-Met, is higher in HNSCC tumors, however, the clinical response rate using these tyrosine kinase inhibitors is limited due to intrinsic and acquired resistance [3]. Therefore, new approaches are necessary to further reduce the mortality of this disease. One approach is to treat HNSCC through dietary means. Natural products are non-toxic and offer promising options for developing effective chemotherapeutics either alone or in combination with existing therapy. Bitter melon (Momordica charantia) is widely cultivated in Asia, Africa, and South America, and extensively used in folk medicines as a remedy for diabetes, specifically in South East Asia. Animal studies have employed either fresh bitter melon extract (BME) or crude organic fractions to evaluate its beneficial effects on glucose metabolism and on plasma and hepatic lipids [4], [5]. We have previously shown that BME (without seeds) treatment of human cancer cells induced cell cycle arrest by altering critical signaling molecules and impairing cell growth [6], [7]. BME feeding also prevented high grade prostatic intraepithelial neoplasia formation in a TRAMP mouse model [7]. Antiproliferative activity of bitter melon extract was recently noted in other cancer cell lines [8]–[10]. However, the anti-cancer potential of BME against HNSCC and its underlying mechanism has not been explored. In this study, we have examined the effect of BME in a number of HNSCC cell lines. We have demonstrated for the first time that BME treatment inhibits c-Met expression and its downstream signaling pathway. Further, we have observed that BME feeding in mice regresses the HNSCC xenograft tumor growth, suggesting its potential as a therapeutic agent against HNSCC.

Materials and Methods Cell Lines Cal27 cells (tongue, mutant p53) were recently obtained from ATCC. JHU-22 (larynx, wild type of p53) and JHU-29 (tongue, wild type of p53) cells were recently procured from the Johns Hopkins University [11], [12]. Cal27 and JHU-22 cells were maintained in Dulbecco's Modified Eagle Medium (Sigma) supplemented with 10% FBS and 1% penicillin/streptomycin (Sigma). JHU-29 cells were maintained in RPMI-1640 Medium (Sigma) supplemented with 10% FBS and 1% penicillin/streptomycin. Cal27, JHU-22 and JHU-29 cells were treated with different doses of BME, and untreated cells were used in parallel as controls. BME was prepared from the Chinese variety of young bitter melons (raw and green) as discussed previously [6]. Briefly, BME was extracted using a household juicer and centrifuged at 560 x g at 4°C for 30 min, freeze dried at -45°C for 72 h and stored at −80°C until used for feeding studies. We prepared a stock of 0.1 g/ml in water, aliquoted, and used for in vitro cell culture work and 100 µl/mouse for oral gavage. Cell proliferation assay Trypan blue exclusion method was used to investigate cell proliferation in control and BME treated Cal27 cells. Live cells were counted using a hemocytometer (Fisher Scientific) at different time points. MTS assay (Promega) was also used for cell viability assay. Human Cell Cycle Array RNA was isolated from control and BME treated Cal27 cells. A RT2 profiler PCR Array for human cell cycle (Qiagen Inc., PAHS-020Z) was performed as described previously [13]. Array data was analyzed using free web based software http://pcrdataanalysis.sabiosciences.com/PCR/arrayanalysis.php and automatically perform all ΔΔC t fold change calculations. Xenograft tumor growth assay Cal27 cells were trypsinized, washed, and resuspended in serum free Dulbecco's Modified Eagle Medium. 2×106 (100 µl) cells containing 40% BD-Matrigel were injected subcutaneously into the flank of five week old BALB/c athymic nude mice (Harlan Laboratories). When tumor volume reached ∼60 mm3, mice were randomly divided in two groups. One group received 100 µl of BME by gavage daily for 5 days/week and the other group received 100 µl of ddH 2 0 by gavage for control, as described previously [7]. BME dosage was selected based on our previous study [7]. Tumors were measured using a slide Caliper once a week and volume was calculated using the formula L x H x W x 0.5236, as described previously [14], [15]. After 4 weeks of treatment, mice were sacrificed; tumors were dissected and divided into two groups. In one group, tumors were fixed in formalin and processed for H & E staining and immunohistochemistry. The other group of tumors was snap frozen for biochemical analysis. Ethics statement The animal experiments are conducted using highest standards for animal care in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals, and approval of Saint Louis University Animal Care Committee (Approval number: 1017). Western Blotting Cell lysates were prepared from control or BME treated Cal27, JHU-22 and JHU-29 cells for Western blot analysis using specific antibodies. Protein lysates were also prepared from collected tumor tissues of control or BME treated Cal27 xenograft mice. Proteins were separated by SDS-PAGE and transferred onto 0.45 µM nitrocellulose membrane. Membranes were blocked using 5% low fat dry milk in TBST and probed with the following primary antibodies. Proteins were detected using ECL Western Blotting Substrate (Thermo Scientific) and autoradiography. Protein loads were normalized using antibodies for GAPDH (Cell Signaling Technologies) or tubulin (Santa Cruz Biotechnology). PCNA expression level was examined from control and BME-fed mice by immunohistochemistry (IHC). The following antibodies were used in this study: c-Met, c-myc, Stat3, phospho-Stat3 (Tyr 705), Mcl-1, cleaved caspases 3 and 9, PARP (Cell Signaling Technologies), and Cyclin D1 (Santa Cruz Biotechnology). Statistical analysis Two-tailed Student's t-test was used for statistical analysis, as described previously [14], [15].

Discussion In this study, we have demonstrated that BME treatment significantly reduces cell proliferation of several human HNSCC cell lines. Aberrant activation of the c-Met pathway has been implicated in the development and progression of head and neck cancer [2], [3], [24], [25]. Numerous antagonists and c-Met inhibitors have been developed to target the HGF/c-Met signaling pathway over recent years [16], [25]. Thus, the c-Met pathway is a potential therapeutic target for HNSCC. Our data suggested that BME treatment in HNSCC cell lines (Cal27 and JHU-22) display significant inhibition of c-Met expression. Subsequent studies demonstrated that BME treatment modulates the downstream molecules of c-Met signaling pathway, such as, phosphoStat3, c-myc, and Mcl-1 in HNSCC cells. However, we did not see significant alteration of EGFR or phosphoAKT expression in BME treated Cal27 cells (data not shown). Apoptosis in response to chemotherapeutic drugs is one of the common mechanisms in cancer cells [26]. We have observed that BME treatment in HNSCC cell lines are inducing apoptotic cell death. Cell cycle progression plays an important part in tumor growth and its regulation is an effective strategy for the control of tumor growth [20]. Our in vitro data indicated that BME treatment of HNSCC cells resulted in downregulation of cyclin D1 and survivin, and upregulation of p21/p27. These data suggested that BME exerts a role in HNSCC cell growth regulation as we observed previously with breast and prostate cancer cells [6], [7]. Together these data suggested that cell cycle regulation is another signaling pathway by which BME exerts anti-tumor effect in HNSCC cells. We have used an in vivo model of tumor xenograft to verify the chemotherapeutic potential of BME against HNSCC cell growth. We have shown previously that BME feeding prevented high grade prostatic intraepithelial neoplasia formation in a TRAMP mouse model without any toxic effect [7]. Here, our results suggested that BME feeding in Cal27 xenograft bearing mice display reduced tumor growth without any apparent sign of toxicity in the athymic nude mice. The identification of molecular targets is important in terms of monitoring the clinical efficacy of cancer therapeutic strategies. Our data strongly demonstrated that upon BME feeding reduced expression of c-Met, c-myc, PCNA and MCM2 as signature molecular targets was observed in Cal27 xenograft tumors. In summary, our results demonstrated for the first time the chemotherapeutic efficacy of BME on head and neck cancer cell growth in vitro and tumor xenograft growth in vivo by inhibiting the c-Met signaling pathway. Thus, BME appears to be an attractive dietary product for the management of head and neck cancer. Future studies are indeed necessary using different in vivo models to further verify efficacy and identify additional molecular targets of BME for inhibition of HNSCC growth.

Acknowledgments The authors thank Dr. Cheri West, Frank Strebeck, and Anna Knobeloch for their assistant with feeding BME to tumor bearing nude mice, and Mona Mirkhaef for immunohistochemistry.

Author Contributions Conceived and designed the experiments: AR MV PN RR. Performed the experiments: AR SS RS PV JGG RR. Analyzed the data: AR SS RS PV JGG SC MV RR. Contributed reagents/materials/analysis tools: PN. Wrote the paper: AR SS RS PV JGG SC MV RR.