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Published in final edited form as: Cancer Prev Res (Phila). 2017 Oct 23;11(4):191–202. doi: 10.1158/1940-6207.CAPR-17-0237

Bitter Melon prevents the development of 4-NQO-induced oral squamous cell carcinoma in an immunocompetent mouse model by modulating immune signalling

Subhayan Sur 1, Robert Steele 1, Rajeev Aurora 2, Mark Varvares 3, Katherine E Schwetye 1, Ratna B Ray 1,3,*
PMCID: PMC5882513  NIHMSID: NIHMS914534  PMID: 29061560

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and tobacco is one of the most common factors for HNSCC of the oral cavity. We have previously observed that bitter melon (Momordica charantia) extract (BME) exerts anti-proliferative activity against several cancers including HNSCC. In this study, we investigated the preventive role of BME in 4-nitroquinoline 1-oxide (4-NQO) carcinogen-induced HNSCC. We observed that BME feeding significantly reduced the incidence of 4-NQO induced oral cancer in a mouse model. Histological analysis suggested control 4-NQO treated mouse tongues showed neoplastic changes ranging from moderate dysplasia to invasive squamous cell carcinoma, whereas no significant dysplasia was observed in the BME-fed mouse tongues. We also examined the global transcriptome changes in normal vs. carcinogen-induced tongue cancer tissues, and following BME feeding. Gene ontology and pathway analyses revealed a signature of biological processes including “immune system process” that is significantly dysregulated in 4-NQO-induced oral cancer. We identified elevated expression of pro-inflammatory genes, s100a9, IL23a, IL-1β and immune check point gene PDCD1/PD1, during oral cancer development. Interestingly, BME treatment significantly reduced their expression. Enhancement of MMP9 (“ossification” pathway) was noted during carcinogenesis, which was reduced in BME-fed mouse tongue tissues. Our study demonstrates the preventive effect of BME in 4-NQO-induced carcinogenesis. Identification of pathways that involved in carcinogen-induced oral cancer provides useful information for prevention strategies. Together, our data strongly suggest the potential clinical benefits of BME as a chemopreventive agent in the control or delay of carcinogen-induced HNSCC development and progression.

Keywords: Bitter melon extract, RNA-Seq analysis, oral cancer, immune system process

Introduction

Head and neck squamous cell carcinoma (HNSCC) represents heterogeneous disease, and oral cavity squamous cell carcinomas including tongue cancers are more common. Oral cancer is often associated with tobacco use, alcohol consumption, an unhealthy diet, an inactive lifestyle and poor oral hygiene. The overall survival rate has not improved in the past several of decades, despite significant improvements in surgical procedures, radiotherapy, and chemotherapy, and several factors that contribute to this poor outcome. Thus, there is unmet need for prevention and additional therapeutic intervention.

The 4-NQO (4-nitroquinoline 1-oxide) oral cancer model allows us to study the initiation and prevention of chemically induced cancers of the oral epithelium in vivo (1, 2). In this model, immunocompetent mice treated with 4-NQO develop invasive squamous cell carcinoma of the oral cavity with near 100% penetrance. This model mimics how chronic tobacco abuse contributes to human oral cancers and therapeutic treatments can reduce or prevent these malignancies (3). 4-NQO is a synthetic, water soluble carcinogen, which mimics the chronic tobacco consumption effect by promoting DNA adduct formation, A-G nucleotide substitution and intracellular oxidative stress, resulting in histological and molecular alterations similar to human oral carcinogenesis (3).

One of the main challenges in cancer therapy is the excessive toxicity of chemotherapeutics due to their nonselective activity. Natural products play a critical role in the discovery and development of numerous drugs for the treatment of various types of deadly diseases, including cancer. Therefore, the use of natural products as preventive medicines is becoming increasingly important. In our previous studies, we showed that bitter melon (Momordica charantia) extract (BME) treatment of human cancer cells induces cell cycle arrest by altering critical signalling molecules and impairing cell growth (4–6). BME feeding also prevented high grade prostatic intraepithelial neoplasia formation in the TRAMP mouse model (5). We recently observed that BME augments NK-cell-mediated HNSCC killing activity and reduces the Th17 cell population in the tumor, implicating an immunomodulatory role in a syngeneic mouse model (7, 8).

In this study, we generated oral/tongue squamous cell carcinoma (OSCC) mouse model by adding 4-NQO to drinking water. To examine the effect of BME in the prevention of OSCC development, we included a group of mice with 4-NQO with BME to drinking water. Our results strongly demonstrated that BME feeding reduced 4-NQO induced OSCC growth. Our results also provide important information about the molecular features of the carcinogenesis and chemoprevention. To our knowledge, this is the first study describing the prevention of carcinogen-induced oral cancer progression by BME in immunocompetent mouse model.

Materials and Methods

Tumor development in the mouse oral cavity

C57BL/6 mice (average body weight 18 gm) were obtained from Charles River (USA) and housed in a specific pathogen-free facility at the Saint Louis University. Mice were maintained at 25 ± 5°C temperature, with alternating 12h light/dark cycle and 45–55% humid conditions. All the animal experiments were carried out in accordance with NIH guidelines, following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Saint Louis University.

For oral cancer development, mice (n= 10) were given 4-NQO (50 μg/ml) in their drinking water for 16 weeks, then only water until the end of the experiment. The other group of mice (n= 10) received BME (30% v/v, 600 mg/mouse) in the drinking water along with 4-NQO treatment for 16 weeks, and then continued with only BME until the end of the experiment. The BME was prepared from the Chinese variety of young bitter melons (raw and green) as described previously (7). Briefly, BME was extracted from bitter melon without seeds using a household juicer, centrifuged at 15000 x g at 4°C for 30 min, and stored at −80 °C. The dose of the BME for animal experimentation was selected based on our previous studies. Experimental design is summarized in Fig 1A. Mice were sacrificed at 22 weeks, tongue tissue was macroscopically examined, and the number of macroscopic lesions was counted. One part of tissue was fixed in formalin for histopathological analysis and other part was snap frozen in liquid nitrogen for biochemical analysis.

Fig. 1. Effect of BME on 4-NQO induced mouse tongue carcinogenesis.

Fig. 1

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A: Schematic representation of experimental design. B: Changes in mice body weights in different weeks during 4-NQO treatment with/ without BME. Data are represented as mean ± SD. Small bar indicates standard error (*, p<0.05, **, p<0.01). C: Total number of macroscopic lesions counted in individual tongues of mice exposed to 4-NQO and mice exposed to 4-NQO + BME-fed group. Small bar indicates standard error (***, p<0.001).

Histopathological analysis

Histological evaluation was performed with formalin-fixed, paraffin- embedded tongue tissues. The tongue sections were stained by hematoxylin and eosin (H&E) and observed under bright field microscopy. Histopathological stages were confirmed by a pathologist in a blinded fashion.

RNA isolation and RNA-Seq analysis

Total tissue RNA was extracted from frozen tongue samples by TRIzol reagent and subjected to Next Generation RNA Sequencing (IlluminaNextSeq 500 1x75) at MOgene, LC. TopHat and BowTie software installed in Illumina BaseSpace was used for assembling contigs. The CLC Genomics Workbench 10.0 software was used for additional data analysis. The reads per kilo base of exon model per million mapped reads (RPKM) were calculated and statistical analysis was performed to determine differential gene expression among different groups. Gene Ontology (GO) mapping was performed using custom scripts. The mapping between reads (ENSEMBL identifiers) and GO identifier codes was downloaded from EBI. GO enrichment was assessed using Fisher’s exact P value using a contingency table of the number of genes that observed to be differentially expressed with a GO category, the total number of transcripts in the GO category, the total number of differentially expressed genes and the total number of GO categories in the RNA-Seq dataset.

mRNA expression analysis

Total RNA (1μg) was used with Super Script III Reverse Transcriptase (Life Technology, USA) following the manufacturer’s protocol for cDNA synthesis. Gene expression of S100a9 (Mm 00656925_m1), IL23a (Mm 00518984_m1), IL1b (Mm 00434228_m1), PDCD1 (Mm 01285676_m1) and MMP9 (Mm 00442991_m1) was carried out by real-time PCR using the Taqman gene expression assay (Thermo Fisher Scientific). The mouse 18s gene was used as an endogenous control and for target gene normalization. The relative gene expression was analysed by using the 2−ΔΔCT method, and relative expression was graphically presented.

Protein extraction and Western blot analysis

Tissue lysates were prepared from the frozen samples by using 2× SDS sample buffer, and Western blot analysis was performed using a specific antibody to proliferating cell nuclear antigen (PCNA) (1:1000, Santa Cruz Biotechnology). The blot was reprobed with GAPDH antibody (Cell Signaling) to compare protein load in each lane. Densitometric analysis was done by using Image J software.

Statistical analysis

Data obtained from the 4-NQO-treated (cancer) group were compared with normal mouse tongue (as control), and data obtained from 4-NQO + BME-fed (experimental) group were compared with cancer group. Statistical analysis was performed using the student’s t-test. P-value < 0.05 was considered as statistically significant. Data were expressed as mean with standard deviation (SD).

Results

Effect of bitter melon extract (BME) on 4-NQO induced mouse oral cancer

Our previous studies demonstrated anti-proliferative and immunomodulatory effects of BME on in-vitro and in-vivo oral cancer models (6–8). In this study, we wanted to examine the preventive effect of BME in 4-NQO induced cancer model, which mimics the tobacco-related oral cancer. For this, mice (C57BL/6) were given 4-NQO with or without BME in the drinking water. The volume of water intake and animal health were closely monitored. We terminated our experiment at the 22nd week, since two mice in 4-NQO treated group had large tumor in the tongue and looked sick, were not eating well and had lost significant weight. Tongue was harvested from each mouse for histological and biochemical analysis. The body weight of the mice was lower in 4-NQO treated group as compared to BME-fed group after 18 weeks (Fig. 1B). Macroscopic lesions were more prominent in 4-NQO treated group as compared to BME-fed group (Fig. 1C). Tongue histology was analysed by a pathologist (K. Schwetye) in a blinded fashion. The number of lesions per mouse was counted, and based on histopathological examination classified as mild to severe dysplasia, or squamous cell carcinoma. Hyperkeratosis, acantholysis, large and abnormal nuclei and tumor infiltrating lymphocytes (TIL) were observed in the 4-NQO treated group. Two mice developed invasive squamous cell carcinoma, while most of the mice displayed severe dysplasia. Interestingly no remarkable histological abnormalities were seen in BME-fed mice, only two mice developed mild dysplasia. Representative histological images are shown in Fig. 2A. We also examined the expression of PCNA in both groups of mouse tongue. Western blot analysis demonstrated that PCNA expression was inhibited in BME-fed mice as compared to 4-NQO treated group (Fig. 2B).

Fig. 2. BME prevents 4-NQO-induced oral cancer.

Fig. 2

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A: Representative photograph of lesions observed in mice treated with 4-NQO with or without BME for 22 weeks (100x). Haematoxylin and eosin (H&E) stained section in 4-NQO treated mouse tongue showed invasive tumor (squamous cell carcinoma). 4-NQO +BME treated mouse tongue histology does not display abnormality. SqEp, normal squamous epithelium; LaPr, lamina propria; SkMu, skeletal muscle; thin arrows, nests of invasive squamous carcinoma invading through lamina propria into skeletal muscle; SaGl, salivary gland (thick arrow). B: Western blot analysis of proliferation marker PCNA in 4-NQO induced tongue lesions with and without BME at 22 week. The blot was reprobed with an antibody to GAPDH as an internal control. Densitometry analyses was performed from three experiments using Image J software and shown on the right. Data are represented as mean ± SD. Small bar indicates standard error (**, p<0.01).

RNA-Seq analysis of 4-NQO induced oral cancer and BME treatment

To understand the global gene expression pattern, RNA-Seq was performed from RNA of normal mice (control-without any treatment), 4-NQO treated mice (cancer group) and mice treated with 4-NQO+ BME (BME-fed experimental group) tongue tissues in biological triplicates. The RNA sequence data generated a set of 22,782 transcripts. The differentially expressed genes was obtained from TopHat/Cufflinks analysis (specifically CuffDiff) as described earlier (9). A P value cutoff of 0.01 was used, to account for expression values across all replicates. Out of annotated 22,782 transcripts (data not shown), differential expression between normal vs. 4-NQO induced tumor, and 4-NQO induced tumor vs. BME-fed experimental group was shown (Fig. 3A and 3B). Among them, 6242 genes were differentially expressed between the 4-NQO treated and normal (control) group, of which 2146 genes were unique (Fig. 3C). On the other hand, 4482 genes were differentially regulated in the experimental (BME and 4-NQO fed group) vs. 4-NQO treated group, where 634 genes are unique (Fig. 3C). Further, 1330 genes were differentially regulated between the experimental and normal (control) group and 466 genes were unique. There were 386 genes commonly altered among the three groups (Fig. 3C). Thus, the data indicates modulation in expression of a huge number of genes during BME-mediated tongue cancer prevention.

Fig. 3. RNA sequence analysis on global gene expression profile during 4-NQO induced mouse tongue carcinogenesis and chemoprevention by BME.

Fig. 3

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Graphical representation of total genes expression significantly increased or decreased (p<0.05) in (A) 4-NQO treated cancer group compared to normal and (B) BME-fed group compared to 4-NQO treated cancer. C: Venn diagram provides a count of genes that were differentially expressed and how they were shared or were unique to each sample type (Normal, cancer and experimental samples).

Gene ontology analysis in 4-NQO-induced oral cancer and BME-fed mice

Next, gene ontology (GO) analysis was performed using custom scripts (see Methods for details) as previously described (10). GO analysis showed significantly (p<0.05) up-regulated GO categories including “keratin filament”, “ion transport”, “structural molecule activity”, “membrane”, “calmodulin binding”, and “regulation of ion transmembrane transport” in the 4-NQO-induced cancer samples as compared to normal group (Fig. 4A). In contrast, “inflammatory response”, “extracellular space”, “cytokine activity”, “immune response”, “structural molecule activity”, and “cell chemotaxis” were significantly downregulated in the 4-NQO-induced cancer group as compared to normal samples (Fig. 4A). BME treatment resulted in significant up-regulation of “extracellular space”, “cytokine activity”, “immune response”, “inflammatory response”, “cell chemotaxis”, and “positive regulation of apoptotic process” (Fig. 4B), and downregulation of “keratin filament”, “extracellular region”, “GTP binding”, and “lipid metabolic process” in the experimental group as compared to the 4-NQO-induced cancer group (Fig. 4B).

Fig. 4. Gene ontology (GO) analysis of the genes in different groups.

Fig. 4

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A: GO analysis of the genes whose mRNA levels were significantly upregulated or significantly downregulated in 4-NQO treated cancer group compared to normal. B: GO analysis of the genes whose mRNA levels were significantly upregulated or significantly downregulated in BME-fed experimental group compared to 4-NQO treated cancer group.

The GO categories, for which deregulated genes were significantly enriched between experimental and cancer groups, were selected. The differentially expressed genes between the groups were categorized under GO of “biological processes” (Fig. 5). For both comparisons, significantly enriched (P= 2.2X10−16) GO categories were “signal transduction (GO:0007165)”, “apoptosis process (GO: 0006915)”, “metabolic process (GO: 0008152)”, “cell adhesion (GO:0007155)”, “lipid metabolism (GO:0006629)”, “immune system process (GO: 0002376)”, “angiogenesis (GO: 0001525)”, “ossification (GO: 0001503)”, and “G1/S transition of mitotic cell cycle (GO: 0000082)” (Fig. 5).

Fig. 5.

Fig. 5

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Some top significantly enriched GO categories (P= 2.2X10−16) under ontology of Biological processes in BME treated group compared to cancer control group.

Comparison of individual transcripts in 4-NQO- induced oral cancer and BME treatment from immune system process

We have shown previously that BME treatment modulates signal transduction pathways and induces apoptotic cells death (6) and current RNA-Seq data is in agreement with our previous results. We also recently observed immunomodulation by BME in a syngeneic HNSCC mouse model. Further, HNSCC has been intensively studied as an immunosuppressive disease (11), and we focussed our analysis here in immune modulation We compared the mRNA expression change of selected individual genes of the significantly enriched immune system processes between the 4-NQO induced cancer group and BME-fed experimental group. RNA-Seq data in this study suggested a significant up-regulation of s100a9 (686.7 fold), IL23a (13.41 fold), IL1b (18.88 fold) and PDCD1 (17.61 fold) in the 4-NQO-induced cancer group as compared to normal mice under “immune system process (GO: 0002376)”. Elevated expression of pro-inflammatory molecules s100a9, IL23a and IL1b has been reported in different cancers including human oral cancer (12–15). Increased expression of immune check point regulatory gene PDCD1 has also been reported in human oral cancer and other cancers (16). Interestingly, the BME-fed group displayed 3.02, 7.57, 5.99 and 1.7 fold downregulation of s100a9, IL23a, IL1b and PD1 genes, respectively. For validation, we examined expression status of these transcripts in 4-NQO induced tongue tissues with and without BME treatment. Similar to the RNA-Seq data, significant up-regulation of s100a9, IL23a, IL1b and PDCD1 were observed in 4-NQO induced cancer tongue tissues as compared to normal tongue tissues (Fig. 6A). This further confirmed that modulation of the immune system might be necessary for progression of in-vivo OSCC. We also observed significant downregulation of these genes in BME-fed mice as compared to 4-NQO-induced cancer group (Fig. 6A), indicating an important mechanism of chemoprevention by BME. String analysis showed association of these genes with each other under the immune system processes (Fig. 6B), suggesting functional importance of this biological process.

Fig. 6. Validation of some gene expression involved in “immune system process (GO: 0002376)” during 4-NQO induced tongue carcinogenesis and prevention by BME.

Fig. 6

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A: Relative mRNA expression of s100a9, IL23a, IL1b and PDCD1 of different groups was analysed by quantitative RT-PCR. Mouse 18S gene was used as endogenous control and for target gene normalization. Data are represented as mean ± SD. Small bar indicates standard error (*, p<0.05). B: String analysis network module showed functional association of differentially expressed genes under GO of “immune system process”. Sky blue lines, known interaction from curated data bases; pink lines, experimentally determined; green lines, predicted interaction of gene neighbourhood; red lines, gene fusion; blue lines, gene co-occurrence. Colored nodes, query proteins and first shell of interactors; white nodes, second shell of interactors.

Under “ossification process” (GO: 0001503), RNA-Seq data showed 6.8 fold up-regulation of MMP9 in 4-NQO induced oral cancer tissues and 8.1 fold downregulation in the BME-fed group as compared to cancer group. Increased MMP9 expression has been reported in human oral cancer (17, 18). A significant up-regulation of MMP9 was observed in 4-NQO-induced cancer tissues; MMP9 was also significantly downregulated in BME-fed group (Fig. 7A). String analysis of ossification process shows the potential functional association of MMP9 and other molecules under this biological process (Fig. 7B).

Fig. 7. Validation of gene expression involved in “ossification (GO: 0001503)” during 4-NQO induced oral carcinogenesis and prevention by BME.

Fig. 7

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Relative mRNA expression of MMP9 in different groups was analyzed by quantitative RT-PCR. The mouse 18S gene was used as endogenous control and for target gene normalization. Data are represented as mean ± SD. Small bar indicates standard error (*, p<0.05). B: String analysis showed possible functional association of MMP9 and other molecules under GO of “ossicification process”. Sky blue lines, known interaction from curated data bases; pink lines, experimentally determined; green lines, predicted interaction of gene neighbourhood; red lines, gene fusion; blue lines, gene co-occurrence. Colored nodes, query proteins and first shell of interactors; white nodes, second shell of interactors.

Discussion

In this study, we observed that BME feeding in the 4-NQO OSCC mouse model significantly reduced the incidence of tongue tumor as compared to 4-NQO treated mice without any apparent sign of toxicity. Histological study revealed that most of the BME-fed mice displayed no histopathologic abnormality. On the other hand, 4-NQO treated mice displayed a range of neoplastic changes, from moderate dysplasia to invasive squamous cell carcinoma. The identification of molecular targets is important in terms of monitoring the clinical efficacy of cancer therapeutic strategies. PCNA plays a crucial role as an integral component of the eukaryotic DNA replication machinery in normal cellular growth and differentiation (19), and is required for cell growth and cell cycle progression in mammalian cells. Our data strongly demonstrated a reduced expression of PCNA in tongue tissues of BME-fed mice as compared to carcinogen only- treated mice. In the United States, head and neck cancer accounts for 3 percent of malignancies, with approximately 63,000 Americans developing head and neck cancer annually and 13,000 dying from the disease (20). Employing chemopreventive strategies will reduce recurrence and/or secondary tumor growth (metastasis), and will have beneficial effect clinically to improve patients’ survival with HNSCC. Our pilot study using limited number of mice suggested that BME feeding with 4-NQO delayed tumor initiation (~11th week of treatment) in mouse tongue (data not shown), suggesting the chemopreventive implication of BME.

We have shown previously that BME treatment in HNSCC inhibited c-Met- phosphoStat3signaling pathway (6). Targeting Stat3 signalling by static in 4NQO induced HNSCC model has been reported earlier (21). Recently, sulforaphane treatment in the same model showed encouraging data of reduced HNSCC by targeting Stat3 targeting (22). Intraperitoneal injection of metformin, a common diabetes drug, also prevented the development of HNSCC (23). Crude extract of bitter melon displayed anticancer effect in several preclinical models with different mechanism (5, 6, 24, 25). There are several components identified as active compounds for BME with limited follow-up studies. We identified cholesteryl β-D-glucopyranoside as an active component of BME in in vitro study (unpublished data). However, efficacy of this compound is not very strong in our in vitro systems as compared to whole BME. In fact, much of the evidence in cancer chemoprevention suggests that bitter melon crude extract (mimicking the whole fruit components) has a stronger effect as compared to fractionated active components. A similar result was reported from black berry extract (26). Further, use of whole plants or their simple extracts is cost-effective and less toxic. Therefore, BME as a chemopreventive agent in controlling carcinogen such as tobacco induced oral cancer is appealing.

Global transcriptome analysis is important to understand the molecular mechanism of carcinogenesis and chemoprevention. Limited information about tobacco-related HNSCC transcriptomes was available, and their modulation following BME feeding remains unknown. We observed differential expression of transcripts in the normal and 4-NQO-induced cancer groups. We also observed alternation of transcripts with BME-fed mice in comparison with the experimental group. We recently reported that BME exerts an immunomodulatory role in an HNSCC syngeneic mouse model (7, 8). In our RNA-Seq data, we observed modulation in “immune response” and “inflammatory response”, in the BME-fed mice. The inflammatory mediators such as cytokine/chemokines present in the tumor microenvironment either promote or inhibit inflammation-mediated tumorigenesis depending on the immune defense mechanism (27). Different non-steroidal anti-inflammatory drugs showed beneficial effects in combination with conventional therapies in the treatment of different cancers (28). Thus, modulation of these pathways may be an important chemoprevention mechanism by BME with respect to oral cancer.

Immunosuppression was noted in HNSCC. We identified important molecules in the “immune system process” from our array data, and validated the expression of the molecules in 4-NQO-induced and BME-fed tongue tissues. We observed significant upregulation of s100a9, IL23a and IL1beta transcripts in 4-NQO-induced cancer tissues. Elevated expression of pro-inflammatory markers s100a9, IL23a and IL1beta in the cancer samples indicated their importance in in-vivo oral carcinogenesis, as reported in human cancers including HNSCC (12–14). However, downregulation of s100a9 was noted recently in oral cancer samples (29). It is possible that s100a9 may have anti- or pro-tumor responses depending on the intracellular milieu. Elevated expression of s100a9 was reported earlier in the 4-NQO induced mouse tongue cancer study (30), in agreement with our observation. Further, pharmaceutical targeting of s100a9 showed an effective result in phase II clinical trial against metastatic prostate cancer (31).

IL-23 is important for the differentiation of Th17 lymphocytes, and its expression depends on tumor microenvironment. We have shown previously that Th17 cell populations were decreased in the HNSCC tumors following BME feeding (7), in agreement with our current data.

In addition, elevated expression of PDCD1/ PD-1 in cancer samples indicated modulation of immune check point regulation during carcinogenesis. Up-regulation of the PD1 receptor was reported in various human cancers, including oral cancer (16). Targeting PDCD1/PD-1 or its signalling pathway showed effective chemotherapeutic effect in phase I- III clinical trials against HNSCC (11). Thus, significant reduction of the pro-inflammatory and immune check-point molecules using bitter melon along with current therapy may have significant preventive effect for recurrence or migration of distant tumor (metastasis).

Recently, Tang et al., (32) reported that pathways of “cell cycle progression” and “disruption of ECM and basement membrane” are activated at an early stage of tongue carcinogenesis. We also observed higher expression of MMP9 in 4-NQO treated group. Interestingly, a significant lower expression of MMP9 was noted when compared with the BME-fed group. MMP9 is involved in different biological processes such as ossification, invasion and metastasis, and its elevated expression was reported in human oral cancer (17, 18). Therefore, BME treatment may play a major role in limiting these processes.

Further, cancer cells frequently show alteration in cellular metabolism (33). Our RNA-Seq data suggested that BME feeding modulates lipid metabolism associated gene expression as compared to 4-NQO-induced OSSC. Different in-vitro and in-vivo studies also reported a potential beneficial effect of bitter melon against lipid and glucose metabolic dysfunction (34). Thus, regulation of metabolism is an important mechanism of BME mediated oral cancer chemoprevention and requites further investigation.

In summary, our study strongly demonstrated that oral feeing of BME prevents the incidence of carcinogen-induced tongue cancer by modulating different biological processes, including those of the immune system. This is the first study clearly indicating transcriptome modulation of different biological process during BME- mediated chemoprevention.

Acknowledgments

Grant Support: This work was supported by research grant R01 DE024942 from the National Institutes of Health (R. B. Ray), and Saint Louis University Cancer Center Seed Grant (R. B. Ray).

We thank Sourav Bhattacharya and Naoshad Muhammad for initial part of this work.

Footnotes

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

References

  • 1.Tang XH, Knudsen B, Bemis D, Tickoo S, Gudas LJ. Oral cavity and esophageal carcinogenesis modeled in carcinogen-treated mice. Clin Cancer Res. 2004;10:301–13. doi: 10.1158/1078-0432.ccr-0999-3. [DOI] [PubMed] [Google Scholar]
  • 2.Vitale-Cross L, Czerninski R, Amornphimoltham P, Patel V, Molinolo AA, Gutkind JS. Chemical carcinogenesis models for evaluating molecular-targeted prevention and treatment of oral cancer. Cancer Prev Res (Phila) 2009;2:419–22. doi: 10.1158/1940-6207.CAPR-09-0058. [DOI] [PubMed] [Google Scholar]
  • 3.Osei-Sarfo K, Tang XH, Urvalek AM, Scognamiglio T, Gudas LJ. The molecular features of tongue epithelium treated with the carcinogen 4-nitroquinoline-1-oxide and alcohol as a model for HNSCC. Carcinogenesis. 2013;34:2673–81. doi: 10.1093/carcin/bgt223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ray RB, Raychoudhuri A, Steele R, Nerurkar P. Bitter melon (Momordicacharantia) extract inhibits breast cancer cell proliferation by modulating cell cycle regulatory genes and promotes apoptosis. Cancer Res. 2010;70:1925–31. doi: 10.1158/0008-5472.CAN-09-3438. [DOI] [PubMed] [Google Scholar]
  • 5.Ru P, Steele R, Nerurkar PV, Phillips N, Ray RB. Bitter melon extract impairs prostate cancer cell-cycle progression and delays prostatic intraepithelial neoplasia in TRAMP model. Cancer Prev Res (Phila) 2011;4:2122–30. doi: 10.1158/1940-6207.CAPR-11-0376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Rajamoorthi A, Shrivastava S, Steele R, Nerurkar P, Gonzalez JG, Crawford S, et al. Bitter melon reduces head and neck squamous cell carcinoma growth by targeting c-Met signaling. PLoS One. 2013;8:e78006. doi: 10.1371/journal.pone.0078006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Bhattacharya S, Muhammad N, Steele R, Peng G, Ray RB. Immunomodulatory role of bitter melon extract in inhibition of head and neck squamous cell carcinoma growth. Oncotarget. 2016;7:33202–9. doi: 10.18632/oncotarget.8898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Bhattacharya S, Muhammad N, Steele R, Kornbluth J, Ray RB. Bitter Melon Enhances Natural Killer-Mediated Toxicity against Head and Neck Cancer Cells. Cancer Prev Res (Phila) 2017;10:337–344. doi: 10.1158/1940-6207.CAPR-17-0046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7:562–78. doi: 10.1038/nprot.2012.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Kiesel J, Miller C, Abu-Amer Y, Aurora R. Systems level analysis of osteoclastogenesis reveals intrinsic and extrinsic regulatory interactions. Dev Dyn. 2007;236:2181–97. doi: 10.1002/dvdy.21206. [DOI] [PubMed] [Google Scholar]
  • 11.Economopoulou P, Perisanidis C, Giotakis EI, Psyrri A. The emerging role of immunotherapy in head and neck squamous cell carcinoma (HNSCC): anti-tumor immunity and clinical applications. Ann Transl Med. 2016;4:173. doi: 10.21037/atm.2016.03.34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Campa D, Hashibe M, Zaridze D, Szeszenia-Dabrowska N, Mates IN, Janout V, Holcatova I, et al. Association of common polymorphisms in inflammatory genes with risk of developing cancers of the upper aerodigestive tract. Cancer Causes Control. 2007;18:449–55. doi: 10.1007/s10552-007-0129-8. [DOI] [PubMed] [Google Scholar]
  • 13.Srikrishna G. S100A8 and S100A9: new insights into their roles in malignancy. J Innate Immun. 2012;4:31–40. doi: 10.1159/000330095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Fang WY, Chen YW, Hsiao JR, Liu CS, Kuo YZ, Wang YC, et al. Elevated S100A9 expression in tumorstroma functions as an early recurrence marker for early-stage oral cancer patients through increased tumor cell invasion, angiogenesis, macrophage recruitment and interleukin-6 production. Oncotarget. 2015;6:28401–24. doi: 10.18632/oncotarget.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Yan Q, Su Y, Zhou Y, Zhu H, Yang X, Xu J. Interleukin-23 strengthens the anti-apoptotic and drug resistance of human tongue squamous cell carcinoma through the Wingless-related integration site/β-catenin pathway. Hua Xi Kou Qiang Yi XueZaZhi. 2015;33:249–54. doi: 10.7518/hxkq.2015.03.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Zandberg DP, Strome SE. The role of the PD-L1:PD-1 pathway in squamous cell carcinoma of the head and neck. Oral Oncol. 2014;50:627–32. doi: 10.1016/j.oraloncology.2014.04.003. [DOI] [PubMed] [Google Scholar]
  • 17.Venugopal A, Uma Maheswari TN. Expression of matrix metalloproteinase-9 in oral potentially malignant disorders: A systematic review. J Oral MaxillofacPathol. 2016;20:474–479. doi: 10.4103/0973-029X.190951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Tanaka T, Imamura T, Yoneda M, Irie A, Ogi H, Nagata M, et al. Enhancement of active MMP release and invasive activity of lymph node metastatic tongue cancer cells by elevated signaling via the TNF-α-TNFR1-NF-κB pathway and a possible involvement of angiopoietin-like 4 in lung metastasis. Int J Oncol. 2016;49:1377–84. doi: 10.3892/ijo.2016.3653. [DOI] [PubMed] [Google Scholar]
  • 19.Wang SC. PCNA: a silent housekeeper or a potential therapeutic target? Trends Pharmacol Sci. 2014;35:178–86. doi: 10.1016/j.tips.2014.02.004. [DOI] [PubMed] [Google Scholar]
  • 20.Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin. 2017;67:7–30. doi: 10.3322/caac.21387. [DOI] [PubMed] [Google Scholar]
  • 21.Peyser ND, Wang L, Zeng Y, Acquafondata M, Freilino M, Li H, et al. STAT3 as a Chemoprevention Target in Carcinogen-Induced Head and Neck Squamous Cell Carcinoma. Cancer Prev Res. 2016;9:657–63. doi: 10.1158/1940-6207.CAPR-16-0089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Bauman JE, Zang Y, Sen M, Li C, Wang L, Egner PA, et al. Prevention of Carcinogen-Induced Oral Cancer by Sulforaphane. Cancer Prev Res. 2016;9:547–57. doi: 10.1158/1940-6207.CAPR-15-0290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Vitale-Cross L, Molinolo AA, Martin D, Younis RH, Maruyama T, Patel V, et al. Metformin prevents the development of oral squamous cell carcinomas from carcinogen-induced premalignant lesions. Cancer Prev Res (Phila) 2012;5:562–73. doi: 10.1158/1940-6207.CAPR-11-0502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kaur M, Deep G, Jain AK, Raina K, Agarwal C, Wempe MF, et al. Bitter melon juice activates cellular energy sensor AMP-activated protein kinase causing apoptotic death of human pancreatic carcinoma cells. Carcinogenesis. 2013;34:1585–92. doi: 10.1093/carcin/bgt081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Muhammad N, Steele R, Isbell TS, Phillips N, Ray RB. Bitter melon extract inhibits breast cancer growth in preclinical model by inducing autophagic cell death. Oncotarget. 2017 doi: 10.18632/oncotarget.19887. (in press) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kresty LA, Mallery SR, Stoner GD. Black raspberries in cancer clinical trials: Past, present and future. J Berry Res. 2016;6:251–261. doi: 10.3233/JBR-160125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Shrihari TG. Dual role of inflammatory mediators in cancer. eCancer. 2017;11:721. doi: 10.3332/ecancer.2017.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Rayburn ER, Ezell SJ, Zhang R. Anti-Inflammatory Agents for Cancer Therapy. Mol Cell Pharmacol. 2009;1:29–43. doi: 10.4255/mcpharmacol.09.05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Khammanivong A, Sorenson BS, Ross KF, Dickerson EB, Hasina R, Lingen MW, et al. Involvement of calprotectin (S100A8/A9) in molecular pathways associated with HNSCC. Oncotarget. 2016;7:14029–47. doi: 10.18632/oncotarget.7373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Mark R, Bermejo JL, Bierhaus A, Plinkert PK, Angel P, Hess J. The receptor for advanced glycation end products is dispensable in a mouse model of oral and esophageal carcinogenesis. HistolHistopathol. 2013;28:1585–94. doi: 10.14670/HH-28.1585. [DOI] [PubMed] [Google Scholar]
  • 31.Markowitz J, Carson WE., 3rd Review of S100A9 biology and its role in cancer. BiochimBiophysActa. 2013;1835:100–9. doi: 10.1016/j.bbcan.2012.10.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Tang XH, Urvalek AM, Osei-Sarfo K, Zhang T, Scognamiglio T, Gudas LJ. Gene expression profiling signatures for the diagnosis and prevention of oral cavity carcinogenesis-genome-wide analysis using RNA-seqtechnology. Oncotarget. 2015;6:24424–35. doi: 10.18632/oncotarget.4420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Luo X, Cheng C, Tan Z, Li N, Tang M, Yang L, et al. Emerging roles of lipid metabolism in cancer metastasis. Mol Cancer. 2017;16:76. doi: 10.1186/s12943-017-0646-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Alam MA, Uddin R, Subhan N, Rahman MM, Jain P, Reza HM. Beneficial role of bitter melon supplementation in obesity and related complications in metabolic syndrome. J Lipids. 2015;2015:496169. doi: 10.1155/2015/496169. [DOI] [PMC free article] [PubMed] [Google Scholar]

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