Mohammed Sikander

Curcumin Nanoformulation for Cervical Cancer Treatment

Cervical cancer is one of the most common cancers among women worldwide. Current standards of care for cervical cancer includes surgery, radiation, and chemotherapy. Conventional chemotherapy fails to elicit therapeutic responses and causes severe systemic toxicity. Thus, developing a natural product based, safe treatment modality would be a highly viable option. Curcumin (CUR) is a well-known natural compound, which exhibits excellent anti-cancer potential by regulating many proliferative, oncogenic, and chemo-resistance associated genes/proteins. However, due to rapid degradation and poor bioavailability, its translational and clinical use has been limited. To improve these clinically relevant parameters, we report a poly(lactic-co-glycolic acid) based curcumin nanoparticle formulation (Nano-CUR). This study demonstrates that in comparison to free CUR, Nano-CUR effectively inhibits cell growth, induces apoptosis, and arrests the cell cycle in cervical cancer cell lines. Nano-CUR treatment modulated entities such as miRNAs, transcription factors, and proteins associated with carcinogenesis. Moreover, Nano-CUR effectively reduced the tumor burden in a pre-clinical orthotopic mouse model of cervical cancer by decreasing oncogenic miRNA-21, suppressing nuclear β-catenin, and abrogating expression of E6/E7 HPV oncoproteins including smoking compound benzo[a]pyrene (BaP) induced E6/E7 and IL-6 expression. These superior pre-clinical data suggest that Nano-CUR may be an effective therapeutic modality for cervical cancer.

Restitution of Tumor Suppressor MicroRNA-145 Using Magnetic Nanoformulation for Pancreatic Cancer Therapy

IntroductionThe functional significance of lost microRNAs has been reported in several human malignancies, including pancreatic cancer (PC). Our prior work has identified microRNA-145 (miR-145) as a tumor suppressor microRNA (miRNA) in pancreatic cancer. The restoration of miR-145 downregulates a number of oncogenes including mucin MUC13, a transmembrane glycoprotein that is aberrantly expressed in pancreatic cancer, thus efficiently inhibiting tumor growth in mice. However, lack of an effective tumor-specific delivery system remains an unmet clinical challenge for successful translation of microRNAs.MethodsWe developed a miRNA-145-based magnetic nanoparticle formulation (miR-145-MNPF) and assessed its anti-cancer efficacy. Physico-chemical characterization (dynamic light scattering (DLS), transmission electron microscopy (TEM) and miR-binding efficiency), cellular internalization (Prussian blue and confocal microscopy), miR-145 restitution potential (quantitative reverse-transcription PCR (qRT-PCR), and anti-cancer efficacy (proliferation, colony formation, cell migration, cell invasion assays) of this formulation were performed using clinically relevant pancreatic cancer cell lines (HPAF-II, AsPC-1).ResultsmiR-145-MNPF exhibited optimal particle size and zeta potential which effectively internalized and restituted miR-145 in pancreatic cancer cells. miR-145 re-expression resulted in downregulation of MUC13, HER2, pAKT, and inhibition of cell proliferation, clonogenicity, migration, and invasion of pancreatic cancer cells.ConclusionsmiR-145-MNPF is an efficient system for miR-145 delivery and restitution in pancreas cancer that may offer a potential therapeutic treatment for PC either alone or in conjunction with conventional treatment.

MUC13 interaction with receptor tyrosine kinase HER2 drives pancreatic ductal adenocarcinoma progression

Although MUC13, a transmembrane mucin, is aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC) and generally correlates with increased expression of HER2, the underlying mechanism remains poorly understood. Herein, we found that MUC13 co-localizes and interacts with HER2 in PDAC cells (reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, co-capping assays) and tissues (immunohistofluorescence). The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at p1248 in PDAC cells, leading to stimulation of HER2 signaling cascade, including ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling and motility, invasion of PDAC cells—all collectively contributing to PDAC progression. Interestingly, all of these phenotypic effects of MUC13–HER2 co-localization could be effectively compromised by depleting MUC13 and mediated by the first and second EGF-like domains of MUC13. Further, MUC13–HER2 co-localization also holds true in PDAC tissues with a strong functional correlation with events contributing to increased degree of disorder and cancer aggressiveness. In brief, findings presented here provide compelling evidence of a functional ramification of MUC13–HER2: this interaction could be potentially exploited for targeted therapeutics in a subset of patients harboring an aggressive form of PDAC.

Cucurbitacin D exhibits potent anti-cancer activity in cervical cancer

In this study, we for the first time, investigated the potential anti-cancer effects of a novel analogue of cucurbitacin (Cucurbitacin D) against cervical cancer in vitro and in vivo. Cucurbitacin D inhibited viability and growth of cervical cancer cells (CaSki and SiHa) in a dose-dependent manner. IC50 of Cucurbitacin D was recorded at 400 nM and 250 nM in CaSki and SiHa cells, respectively. Induction of apoptosis was observed in Cucurbitacin D treated cervical cancer cells as measured by enhanced Annexin V staining and cleavage in PARP protein. Cucurbitacin D treatment of cervical cancer cells arrested the cell cycle in G1/S phase, inhibited constitutive expression of E6, Cyclin D1, CDK4, pRb, and Rb and induced the protein levels of p21 and p27. Cucurbitacin D also inhibited phosphorylation of STAT3 at Ser727 and Tyr705 residues as well as its downstream target genes c-Myc, and MMP9. Cucurbitacin D enhanced the expression of tumor suppressor microRNAs (miR-145, miRNA-143, and miRNA34a) in cervical cancer cells. Cucurbitacin D treatment (1 mg/kg body weight) effectively inhibited growth of cervical cancer cells derived orthotopic xenograft tumors in athymic nude mice. These results demonstrate the potential therapeutic efficacy of Cucurbitacin D against cervical cancer.

Ormeloxifene suppresses prostate tumor growth and metastatic phenotypes via inhibition of oncogenic β-catenin signaling and EMT progression

Ormeloxifene is a clinically approved selective estrogen receptor modulator, which has also shown excellent anticancer activity, thus it can be an ideal repurposing pharmacophore. Herein, we report therapeutic effects of ormeloxifene on prostate cancer and elucidate a novel molecular mechanism of its anticancer activity. Ormeloxifene treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), β-catenin/TCF-4 transcriptional activity, and induced the expression of pGSK3β. In molecular docking analysis, ormeloxifene showed proficient docking with β-catenin and GSK3β. In addition, ormeloxifene induced apoptosis, inhibited growth and metastatic potential of prostate cancer cells and arrested cell cycle in G0–G1 phase via modulation of cell-cycle regulatory proteins (inhibition of Mcl-1, cyclin D1, and CDK4 and induction of p21 and p27). In functional assays, ormeloxifene remarkably reduced tumorigenic, migratory, and invasive potential of prostate cancer cells. In addition, ormeloxifene treatment significantly (P < 0.01) regressed the prostate tumor growth in the xenograft mouse model while administered through intraperitoneal route (250 μg/mouse, three times a week). These molecular effects of ormeloxifene were also observed in excised tumor tissues as shown by immunohistochemistry analysis. Our results, for the first time, demonstrate repurposing potential of ormeloxifene as an anticancer drug for the treatment of advanced stage metastatic prostate cancer through a novel molecular mechanism involving β-catenin and EMT pathway. Mol Cancer Ther; 16(10); 2267–80. ©2017 AACR.

Abstract 3224: Cucurbitacin D inhibits prostate tumor growth via targeting glucose metabolism

Background: Emergence of hormone-refractory PrCa (HRPC) after the anti-androgen therapy, cancer metastasis and chemo-resistance are the major hurdle for the treatment of prostate cancer (PrCa) patients. Accumulative evidence suggests that altered glucose metabolism is one of the mechanisms for metastatic PrCa cell survival and chemo-resistance. Therefore, identification and generation of natural/or synthetic pharmacological agents that can limit altered glucose metabolism might be highly useful for the treatment of metastatic and chemo-resistant PrCa. Cucurbitacins have shown potent anti-cancer and glucagonostic activities along with severe liver toxicity. Thus, a focus on developing different analogues of cucurbitacin is being pursued by scientific community. Herein, we report that Cucurbitacin D, an analogue of cucurbitacin, suppresses the growth of metastatic PrCa cells in vitro and in vivo via targeting glucose metabolism and its associated molecular targets. Methods: HRPC cells (PC3 and DU145) was used as a model system. Effect of Cucurbitacin D on PrCa cell proliferation and apoptosis was performed by MTS, xCELLigence and Annexin V assays. Effect of Cucurbitacin D on clonogenic potential of PrCa cells was examined by colony formation assay. In silico analysis was performed to study if Cucurbitacin D interacts with GLUT1 receptor. Effect of Cucurbitacin D on glucose metabolism of PrCa cells was performed by metabolic shift, glucose and lactate uptake assays. Effect of Cucurbitacin D treatment on key molecules of cell survival and glucose metabolism signaling pathways in PrCa cells was analyzed by Western blot and qRT-PCR analyses. Therapeutic efficacy of Cucurbitacin D against PrCa was evaluated in an ectopic PrCa xenograft mouse model. Results: Cucurbitacin D (0.1 to 1 µM) treatment significantly (P -1 ). It has been reported that miR-132 targets the GLUT1, interestingly, Cucurbitacin D replenished the expression of miR-132 in PrCa cells. These growth inhibitory effects of Cucurbitacin D were confirmed in PrCa xenograft mouse model while administered intra-tumorally (1 mg/kg body weight thrice/week). Conclusion: Our results suggest that Cucurbitacin D is a novel modulator of glucose metabolism which could be a promising therapeutic modality alone or in combination of conventional chemotherapeutic agents for PrCa. Citation Format: Mohammed Sikander, Bilal Bin Hafeez, Shabnam Malik, Aditya Ganju, Fathi T. Halaweish, Murali Mohan Yallapu, Subhash C. Chauhan, Meena Jaggi. Cucurbitacin D inhibits prostate tumor growth via targeting glucose metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3224. doi:10.1158/1538-7445.AM2017-3224

Abstract 1071: Mir-145 based magnetic nanoformulation for pancreatic cancer therapy

Background: Pancreatic cancer (PanCa) is the fourth leading cause of cancer related deaths in the USA, with a 5-year survival rate of less than 5%. MicroRNAs have been identified as attractive targets for therapeutic intervention. The functional significance of lost microRNAs have been reported in several human malignancies, including PanCa. Therefore, restoring lost miRNA function can provide a potential therapeutic benefit. Prior work has identified microRNA-145 (miR-145) as a tumor suppressor miRNA in pancreatic cancer. The restoration of miR-145 downregulates a number of oncogenes including mucin MUC13, a glycoprotein that is aberrantly expressed in PanCa, and efficiently inhibits tumor growth in mice. The main challenge for successful translation of microRNAs into clinical practice remains an effective in vivo delivery system. The focus of this study was to develop and assess the efficacy of a miR-145 based nanoparticle formulation for PanCa treatment. Methods: Magnetic nanoparticle (MNP) based nanoformulation of miR-145 (miR-145-MNPF) was developed for the intracellular delivery and sustained release of miR-145. The positively charged polyethyleneimine molecules were used to increase the loading efficiency of miR-145. MUC13 expressing pancreatic ductal adenocarcinoma cell lines (HPAF-II and AsPC-1) were used for the study. Following transfection of miR-145-MNPF, Western blotting and immunofluorescence techniques were used to investigate the effects of miR-145 restoration on number of proteins including MUC13. Additionally, functional studies of the effects of miR-145 restitution using miR-145-MNPF included cell proliferation, colony formation, cell migration, and cell invasion assays. Results: miR-145 expression was progressively suppressed over the course of development from PanIN I-III to late stage poorly differentiated PDAC. Treatment of cells with miR-145-MNPF led to efficient intracellular delivery of miR-145 mimics as observed through prussian blue staining. This led to the simultaneous upregulation of miR-145 levels in cells as confirmed by qRT-PCR. miR-145 restitution resulted in significant downregulation of target oncogenes including MUC13, HER2, P-AKT and p53 as observed through Western blotting and immunofluorescence techniques. miR-145-MNPF inhibited cell proliferation, clonogenicity, migration, and invasion of PanCa cells. MNPF mediated restitution of miR-145 effectively sensitizes PanCa cells for paclitaxel and TRAIL therapy. Conclusions: 1) MNP based delivery systems can be efficiently used for microRNA replacement therapy in order to restore lost microRNAs in cancer. 2) miR-145-MNPF efficiently restores miR-145 in pancreatic cancer cells and inhibits growth and invasion of PanCa. 3) miR-145 restitution using miR-145-MNPF may offer a potential therapeutic strategy for pancreatic cancer treatment alone or in combination with other therapies. Citation Format: Saini Setua, Sheema Khan, Murali Mohan Yallapu, Mohammed Sikander, Stephen W. Behrman, Meena Jaggi, Subhash C. Chauhan. Mir-145 based magnetic nanoformulation for pancreatic cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1071.

Abstract 3081: Novel cucurbitacin analogue Cuc D exhibits potent anti-cancer activity in cervical cancer

Background: Cervical cancer is one of the leading cause of mortality among women in US. Naturally occurring dietary compounds have gained increasing attention for their anticancer effects. Cucurbitacins, tetracyclic triterpenoid compound, belong to a family of Cucurbitaceae have shown promising anti-cancer activity. Herein, we investigated the potential anti-cancer effects of a novel analogue of cucurbitacin D (Cuc D) against cervical cancer in in vitro and in a xenograft mouse model. Methods: In our study, we used human cervical cancer cells (CaSki and SiHa). Cells were treated with Cuc D (0.05 to 1μM) for 48 and 72 hrs. MTS and colony formation assays were performed to investigate the effects of Cuc D on cell viability and proliferation. Western Blot analysis was performed to investigate the effects of Cuc D on cell proliferation and apoptotic markers. To determine the therapeutic effects of Cuc D, we used female athymic nude mice and injected CaSki cells (4 × 106) into the cervix to develop orthotopic xenograft tumors. Cuc D (1 mg/kg body weight) was administered through intratumoral injection four weeks post-tumor cell injection. Tumor volume in these mice were recorded bi-weekly. Results: Cuc D inhibited cell viability of cervical cancer cells in a dose-dependent manner. IC50 of Cuc D was observed 400 nM and 250 nM in Caski and SiHa cells, respectively. Cuc D treatment effectively inhibited growth of cervical cancer cells which was determined by decreased cell proliferation and colony formation assays. Cuc D treatment induced apoptosis in cervical cancer cells as measured by enhanced Annexin V staining. Western blot result also illustrated cleavage in PARP protein in Cuc D treated cells which further confirms apoptosis induction. Cuc D treatment also inhibited PI3K and c-Myc protein levels and phosphorylation of STAT3 and Rb proteins. In addition, Cuc D treatment induced the cell cycle inhibitory proteins (p21 and p27) and PTEN and the expression of a tumor suppressor microRNA, miR-145, as determined by qRT-PCR. In an orthotopic tumor xenograft mouse model, Cuc D treatment effectively inhibited tumor growth as compared to vehicle control treated mice. Conclusion: Taken together, our results demonstrate potent anti-cancer efficacy of Cuc D in cervical cancer cells via modulation of key onco/tumor suppressor proteins. Thus, Cuc D could be a useful therapeutic agent for cervical cancer treatment. Citation Format: Mohammed Sikander, Bilal Bin Hafeez, Fathi T. Halaweish, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Novel cucurbitacin analogue Cuc D exhibits potent anti-cancer activity in cervical cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3081.

Abstract 182: Smoking Carcinogen (BaP) enhances tumorigenic phenotypes of cervical cancer cells by modulation of HPV oncogenes and microRNA profiles

Cervical cancer (CxCa), one of the most common and deadly cancers among women worldwide, is associated with persistent Human Papillomavirus (HPV) infection. An importunate oncogenic HPV infection itself is not enough to immortalize and transform the epithelial host cell. Additional factors are needed to acquire an immortal, malignant and invasive phenotype. In addition to HPV infection, cigarette smoking is a known risk factor. Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, is among the numerous carcinogens associated with cigarette smoking. BaP levels have been found to be elevated in the cervical mucus of women who smoke. BaP has been shown to stimulate high levels of the viral oncoproteins E6 and E7 besides enhancing virion synthesis in cell lines prolifically infected with HPV. Furthermore, mixtures containing BaP, such as cigarette smoke condensate, have been shown to induce dramatic microRNA (miR) alterations in rodent lungs and in vitro human bronchial models. MiRs, small non-coding RNAs, regulate gene expression and their aberrant regulation/function has been reported in a large majority of cancers. Additionally, previous studies have shown the role of BaP in modulating miR profiles in different cancers. In this study we examine the effects of the carcinogen BaP on miRs and their related pathways in CxCa. In vitro treatment of CxCa cells with BaP resulted in an increase of expression of oncogenic miR-21 and decrease in tumor suppressor miR-214. MiR-21 has been shown to enhance chemoresistance to established regimens of chemotherapy and radiation therapy. In addition BaP also modulates the downstream pathways that these miRs regulate. BaP treatment increased the expression of oncogenic transcription factors such as NFkB-65, pSTAT-3, pSTAT-5 (transcription factors promoting miR-21 expression) and oncogenic cytokine IL-6, besides decreasing tumor suppressor PTEN (a target of miR-21). IL-6 is known to increase miR-21 expression through pSTAT-3 and pSTAT-5. Moreover, BaP in combination with IL-6 enhanced the migratory properties of CxCa cells. BaP also increased the nuclear translocation of β-catenin as observed through confocal microscopy. β-catenin is a direct target of miR-214. Natural or synthetic compounds that can mitigate BaP induced tumorigenic effects can have tremendous clinical significance. Herein, we report that curcumin (C) or its PLGA (poly [lactic-co-glycolic acid]) based nanoformulation (NC) effectively suppresses BaP induced tumorigenicity, regulates the expression of miRs and nuclear translocation of β-catenin associated with BaP in Caski/SiHa CxCa cells. In vivo experiments show that C/NC efficaciously reduced the tumor burden in xenograft mouse model. These data suggest a potential role of smoking in CxCa progression via regulating miR profiles and C/NC may effectively attenuate smoking induced CxCa progression. Citation Format: Mohd Saif Zaman, Neeraj Chauhan, Rishi K. Gara, Diane Maher, Sonam Kumari, Mohammed Sikander, Sheema Khan, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Smoking Carcinogen (BaP) enhances tumorigenic phenotypes of cervical cancer cells by modulation of HPV oncogenes and microRNA profiles. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 182. doi:10.1158/1538-7445.AM2015-182

Abstract 4468: Anticancer activity of novel cucurbitacin analogue in pancreatic cancer

Background: Human pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States. Accumulating studies have witnessed the malfunction of many chemotherapeutic regimens and the current standard-of-care therapy, gemcitabine (GEM), enhances patient survival by only few months. Cucurbitacins, naturally occurring dietary tetracyclic triterpenoid compounds, have shown promising anti-cancer activities. Herein, we investigated the potency and anti-cancer efficacy of a novel analogue of cucurbitacin (Cuc D) in pancreatic cancer cell lines and in a xenograft mouse model. Additionally, we determined efficacy of this analogue on MicroRNAs (miRNAs) which are important regulators of genes that have crucial roles in pancreatic tumorigenesis. Methods: The effect of Cuc D on the growth of pancreatic cancer cells was determined by cell proliferation assay using six pancreatic cancer (MiaPaCa-2, CaPan-1, HPAF-II, Panc-1, BxPC-3 and AsPc-1) cells. Cell growth kinetic assay was carried out at 24, 48, 72 and 96 h. The clonogenic potential of cancer cells was also studied using the colony formation assay. Tumor suppressor miR-145, which is downregulated in pancreatic cancer, directly target MUC13. Thus the effect of Cuc D was also investigated on the expression of miR-145 through qPCR analysis. Immunoblotting techniques were performed to study the known direct targets of miRNA-145 and its associated proteins. The anti-cancer potential of Cuc D in pancreatic cancer was also evaluated in vivo using a xenograft mouse model. Results: Our results demonstrate potent anticancer effects of Cuc D on pancreatic cancer cells. Cuc D induces dose and time dependent inhibition of cell proliferation in a panel of gemcitabine sensitive/resistant pancreatic cancer cell line models at nanomolar concentrations (100-500 nM). It also inhibits colony formation and invasiveness of pancreatic cancer cells. Furthermore, Cuc D blocks the cell cycle progression in G2/M phase and decreases the mitochondrial membrane potential in pancreatic cancer cells. Notably, Cuc D significantly increases the expression of tumor suppressor miR-145 in HPAF-II cells as observed by qPCR. Furthermore, Cuc D decreases the expression of MUC13 and its associated proteins including pAKT and HER2. In addition, it restores the expression of p53 level as studied by immunofluorescence technique. The expression of key oncogenic proteins including NF-κB, STAT3 (Tyr-705) and Mcl-1 were also downregulated. The levels of PTEN and p27 (Kip1) tumor suppressor genes were increased after Cuc D treatment. Additionally, in vivo administration of Cuc D effectively inhibited pancreatic tumor growth in xenograft mouse model. Conclusion: Overall, this study suggests that Cuc D modulates the expression of key oncogenes and tumor suppressors, thus it can be a promising therapeutic modality for pancreatic cancer prevention and treatment. Citation Format: Mohammed Sikander, Sheema Khan, Neeraj Chauhan, Mohd Saif Zaman, Murali Mohan Yallapu, Fathi T. Halaweish, Bhavin Chauhan, Shabnam Malik, Meena Jaggi, Subhash C. Chauhan. Anticancer activity of novel cucurbitacin analogue in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2015-4468