Vasudha Sundram

Curcumin induces chemo/radio-sensitization in ovarian cancer cells and curcumin nanoparticles inhibit ovarian cancer cell growth

BackgroundChemo/radio-resistance is a major obstacle in treating advanced ovarian cancer. The efficacy of current treatments may be improved by increasing the sensitivity of cancer cells to chemo/radiation therapies. Curcumin is a naturally occurring compound with anti-cancer activity in multiple cancers; however, its chemo/radio-sensitizing potential is not well studied in ovarian cancer. Herein, we demonstrate the effectiveness of a curcumin pre-treatment strategy for chemo/radio-sensitizing cisplatin resistant ovarian cancer cells. To improve the efficacy and specificity of curcumin induced chemo/radio sensitization, we developed a curcumin nanoparticle formulation conjugated with a monoclonal antibody specific for cancer cells.MethodsCisplatin resistant A2780CP ovarian cancer cells were pre-treated with curcumin followed by exposure to cisplatin or radiation and the effect on cell growth was determined by MTS and colony formation assays. The effect of curcumin pre-treatment on the expression of apoptosis related proteins and β-catenin was determined by Western blotting or Flow Cytometry. A luciferase reporter assay was used to determine the effect of curcumin on β-catenin transcription activity. The poly(lactic acid-co-glycolic acid) (PLGA) nanoparticle formulation of curcumin (Nano-CUR) was developed by a modified nano-precipitation method and physico-chemical characterization was performed by transmission electron microscopy and dynamic light scattering methods.ResultsCurcumin pre-treatment considerably reduced the dose of cisplatin and radiation required to inhibit the growth of cisplatin resistant ovarian cancer cells. During the 6 hr pre-treatment, curcumin down regulated the expression of Bcl-XL and Mcl-1 pro-survival proteins. Curcumin pre-treatment followed by exposure to low doses of cisplatin increased apoptosis as indicated by annexin V staining and cleavage of caspase 9 and PARP. Additionally, curcumin pre-treatment lowered β-catenin expression and transcriptional activity. Nano-CUR was successfully generated and physico-chemical characterization of Nano-CUR indicated an average particle size of ~70 nm, steady and prolonged release of curcumin, antibody conjugation capability and effective inhibition of ovarian cancer cell growth.ConclusionCurcumin pre-treatment enhances chemo/radio-sensitization in A2780CP ovarian cancer cells through multiple molecular mechanisms. Therefore, curcumin pre-treatment may effectively improve ovarian cancer therapeutics. A targeted PLGA nanoparticle formulation of curcumin is feasible and may improve the in vivo therapeutic efficacy of curcumin.

Anti-cancer activity of curcumin loaded nanoparticles in prostate cancer.

Prostate cancer is the most commonly diagnosed cancer disease in men in the Unites States and its management remains a challenge in everyday oncology practice. Thus, advanced therapeutic strategies are required to treat prostate cancer patients. Curcumin (CUR) is a promising anticancer agent for various cancer types. The objective of this study was to evaluate therapeutic potential of novel poly(lactic-co-glycolic acid)- CUR nanoparticles (PLGA-CUR NPs) for prostate cancer treatment. Our results indicate that PLGA-CUR NPs efficiently internalize in prostate cancer cells and release biologically active CUR in cytosolic compartment of cells for effective therapeutic activity. Cell proliferation (MTS), clonogenic, and Western blot analyses reveal that PLGA-CUR NPs can effectively inhibit proliferation and colony formation ability of prostate cancer cells than free CUR. PLGA-CUR NPs showed superior tumor regression compared to CUR in xenograft mice. Further investigations reveal that PLGA-CUR NPs inhibit nuclear β-catenin and AR expression in cells and in tumor xenograft tissues. It also suppresses STAT3 and AKT phosphorylation and leads to apoptosis via inhibition of key anti-apoptotic proteins, Mcl-1, Bcl-xL and caused induction of PARP cleavage. Additionally, significant downregulation of oncogenic miR21 and up-regulation of miR-205 was observed with PLGA-CUR NPs treatment as determined by RT-PCR and in situ hybridization analyses. A superior anti-cancer potential was attained with PSMA antibody conjugated PLGA-CUR NPs in prostate cancer cells and a significant tumor targeting of (131)I labeled PSMA antibody was achieved with PLGA-CUR NPs in prostate cancer xenograft mice model. In conclusion, PLGA-CUR NPs can significantly accumulate and exhibit superior anticancer activity in prostate cancer.

Novel Curcumin-Loaded Magnetic Nanoparticles for Pancreatic Cancer Treatment

Curcumin (CUR), a naturally occurring polyphenol derived from the root of Curcuma longa, has showed potent anticancer and cancer prevention activity in a variety of cancers. However, the clinical translation of CUR has been significantly hampered due to its extensive degradation, suboptimal pharmacokinetics, and poor bioavailability. To address these clinically relevant issues, we have developed a novel CUR-loaded magnetic nanoparticle (MNP-CUR) formulation. Herein, we have evaluated the in vitro and in vivo therapeutic efficacy of this novel MNP-CUR formulation in pancreatic cancer. Human pancreatic cancer cells (HPAF-II and Panc-1) exhibited efficient internalization of the MNP-CUR formulation in a dose-dependent manner. As a result, the MNP-CUR formulation effectively inhibited growth of HPAF-II and Panc-1 cells in cell proliferation and colony formation assays. The MNP-CUR formulation suppressed pancreatic tumor growth in an HPAF-II xenograft mouse model and improved the survival of mice by delaying tumor growth. The growth-inhibitory effect of MNP-CUR formulation correlated with the suppression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-extra large (Bcl-xL), induced myeloid leukemia cell differentiation protein (Mcl-1), cell surface–associated Mucin 1 (MUC1), collagen I, and enhanced membrane β-catenin expression. MNP-CUR formulation did not show any sign of hemotoxicity and was stable after incubation with human serum proteins. In addition, the MNP-CUR formulation improved serum bioavailability of CUR in mice up to 2.5-fold as compared with free CUR. Biodistribution studies show that a significant amount of MNP-CUR formulation was able to reach the pancreatic xenograft tumor(s), which suggests its clinical translational potential. In conclusion, this study suggests that our novel MNP-CUR formulation can be valuable for the treatment of pancreatic cancer. Mol Cancer Ther; 12(8); 1471–80. ©2013 AACR.

Curcumin Attenuates β-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1

Prostate cancer is the most commonly diagnosed cancer affecting 1 in 6 males in the US. Understanding the molecular basis of prostate cancer progression can serve as a tool for early diagnosis and development of novel treatment strategies for this disease. Protein Kinase D1 (PKD1) is a multifunctional kinase that is highly expressed in normal prostate. The decreased expression of PKD1 has been associated with the progression of prostate cancer. Therefore, synthetic or natural products that regulate this signaling pathway can serve as novel therapeutic modalities for prostate cancer prevention and treatment. Curcumin, the active ingredient of turmeric, has shown anti-cancer properties via modulation of a number of different molecular pathways. Herein, we have demonstrated that curcumin activates PKD1, resulting in changes in β-catenin signaling by inhibiting nuclear β-catenin transcription activity and enhancing the levels of membrane β-catenin in prostate cancer cells. Modulation of these cellular events by curcumin correlated with decreased cell proliferation, colony formation and cell motility and enhanced cell-cell aggregation in prostate cancer cells. In addition, we have also revealed that inhibition of cell motility by curcumin is mediated by decreasing the levels of active cofilin, a downstream target of PKD1. The potent anti-cancer effects of curcumin in vitro were also reflected in a prostate cancer xenograft mouse model. The in vivo inhibition of tumor growth also correlated with enhanced membrane localization of β-catenin. Overall, our findings herein have revealed a novel molecular mechanism of curcumin action via the activation of PKD1 in prostate cancer cells.

Emerging roles of protein kinase D1 in cancer.

Protein kinase D1 (PKD1) is a serine-threonine kinase that regulates various functions within the cell, including cell proliferation, apoptosis, adhesion, and cell motility. In normal cells, this protein plays key roles in multiple signaling pathways by relaying information from the extracellular environment and/or upstream kinases and converting them into a regulated intracellular response. The aberrant expression of PKD1 is associated with enhanced cancer phenotypes, such as deregulated cell proliferation, survival, motility, and epithelial mesenchymal transition. In this review, we summarize the structural and functional aspects of PKD1 and highlight the pathobiological roles of this kinase in cancer. Mol Cancer Res; 9(8); 985–96. ©2011 AACR.

Increased Expression and Aberrant Localization of Mucin 13 in Metastatic Colon Cancer

MUC13 is a newly identified transmembrane mucin. Although MUC13 is known to be overexpressed in ovarian and gastric cancers, limited information is available regarding the expression of MUC13 in metastatic colon cancer. Herein, we investigated the expression profile of MUC13 in colon cancer using a novel anti-MUC13 monoclonal antibody (MAb, clone ppz0020) by immunohistochemical (IHC) analysis. A cohort of colon cancer samples and tissue microarrays containing adjacent normal, non-metastatic colon cancer, metastatic colon cancer, and liver metastasis tissues was used in this study to investigate the expression pattern of MUC13. IHC analysis revealed significantly higher (p<0.001) MUC13 expression in non-metastatic colon cancer samples compared with faint or very low expression in adjacent normal tissues. Interestingly, metastatic colon cancer and liver metastasis tissue samples demonstrated significantly (p<0.05) higher cytoplasmic and nuclear MUC13 expression compared with non-metastatic colon cancer and adjacent normal colon samples. Moreover, cytoplasmic and nuclear MUC13 expression correlated with larger and poorly differentiated tumors. Four of six tested colon cancer cell lines also expressed MUC13 at RNA and protein levels. These studies demonstrate a significant increase in MUC13 expression in metastatic colon cancer and suggest a correlation between aberrant MUC13 localization (cytoplasmic and nuclear expression) and metastatic colon cancer.

Anti-Cancer Potential of a Novel SERM Ormeloxifene

Ormeloxifene is a non-steroidal Selective Estrogen Receptor Modulator (SERM) that is used as an oral contraceptive. Recent studies have shown its potent anti-cancer activities in breast, head and neck, and chronic myeloid leukemia cells. Several in vivo and clinical studies have reported that ormeloxifene possesses an excellent therapeutic index and has been well-tolerated, without any haematological, biochemical or histopathological toxicity, even with chronic administration. A reasonably long period of time and an enormous financial commitment are required to develop a lead compound into a clinically approved anti-cancer drug. For these reasons and to circumvent these obstacles, ormeloxifene is a promising candidate on a fast track for the development or repurposing established drugs as anti-cancer agents for cancer treatment. The current review summarizes recent findings on ormeloxifene as an anti-cancer agent and future prospects of this clinically safe pharmacophore.

Abstract 1032: Ormeloxifene attenuates metastatic and glycolytic pathways in breast cancer cells

Background: Breast cancer is the second most deadly cancer in female, resulting in over 39,000 deaths in year 2012. Triple negative breast cancer is a subtype of breast cancer characterized by its poor outcome and a lack of targeted therapies. New targeted therapies are therefore an urgent unmet medical need for this patient population. Recent investigations into anti-cancer drug are targeted on repurposing pre-approved and safe drugs for cancer treatment. We report for the first time that a non-steroidal, selective estrogen receptor inhibitor, ormeloxifene (ORM) inhibits Metastasis-associated protein 1 (MTA1) and glycolytic pathway through AMPKα 1/2 inhibition in triple negative breast cancer cells. They are both involved in promoting cancer cell survival, proliferation and metastasis. MTA1 is an initiator of epithelial to mesenchymal transition (EMT) which is responsible for metastasis and poor prognosis. AMPKα 2 is important for glycolysis and promotes tumor survival. Methods: Herein, we investigated the use of ORM for the treatment of breast cancer. We used MB-231 and MCF-7 breast cell lines as prototypical model and investigated the effect of ORM treatment on MTA1 and AMPKα 2 expression pattern. We investigated the effect of ORM on cell proliferation (MTS), clonogenic potential (anchorage dependent growth) of the cells and analyzed its effect on markers of EMT through Western blotting. Further, we performed glycolytic function analysis using Seahorse analyzer and mitochondrial membrane potential analysis by Tetramethylrhodamine, ethyl ester (TMRE). We performed Western blotting to investigate the role of ORM on MTA1 and AMPKα inhibition. Results: Our results suggest that ORM inhibits proliferation of both MB-231 and MCF-7 breast cancer cells at low concentrations. Investigation of the EMT marker profile revealed a dose dependent decrease in the expression of MTA1 in both the cells on ORM treatment. This leads to inhibition of motility and invasion of cells in a concentration dependent manner. Additionally, treatment of MB-231 cells with ORM suppresses vimentin expression, a marker for EMT that aids in rapid metastasis. ORM inhibits transcription factors, Snail and Slug, known to promote epithelial-mesenchymal transitions during tumor development. Importantly, it was observed that ORM also inhibits glycolytic pathway in breast cancer cells independent of mitochondrial membrane depolarization. ORM inhibits total AMPKα and dephosphorylates AMPKα 2 in breast cancer cells. Conclusions: Our findings suggest that ORM effectively targets MTA1 and glycolytic pathway and inhibits breast cancer cell growth and EMT. Overall, this study suggests that ORM can be a potential therapeutic modality for triple negative breast cancer treatment. Citation Format: Aditya Ganju, Vasudha Sundram, Keith Miskimins, Rishi Gara, Sheema Khan, Man Mohan Singh, Subhash C. Chauhan, Meena Jaggi. Ormeloxifene attenuates metastatic and glycolytic pathways in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1032. doi:10.1158/1538-7445.AM2014-1032

Abstract 4503: Novel curcumin loaded magnetic nanoparticles for pancreatic cancer treatment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Curcumin (CUR), a naturally occurring polyphenol derived from the root of Curcuma longa, has demonstrated potent anti-cancer and cancer prevention activity in a variety of cancers. However, the clinical translation of curcumin has been significantly hampered due its extensive degradation, suboptimal pharmacokinetics and poor bioavailability. To address these clinically relevant issues, we have developed a novel curcumin loaded magnetic nanoparticle (MNP-CUR) formulation. Herein, we have evaluated the in vitro and in vivo therapeutic efficacy of this novel MNP-CUR formulation in pancreatic cancer. Human pancreatic cancer cells (HPAFII and Panc-1) exhibited efficient internalization of the MNP-CUR formulation in a dose dependent manner. As a result, MNP-CUR formulation effectively inhibited growth of HPAFII and Panc-1 cells in cell proliferation and colony formation assays. Similarly, the MNP-CUR formulation suppressed pancreatic tumor growth in an HPAFII xenograft mice model and improved mice survival by delaying tumor growth. The growth inhibitory effect of MNP-CUR formulation was correlated with the suppression of PCNA, Bcl-xL, Mcl-1, MUC1, collagen I and enhanced membrane β-catenin expression. Interestingly, our MNP-CUR formulation did not show any sign of hemotoxicity and stable after incubation with human serum proteins. Additionally, our MNP-CUR formulation improved serum bioavailability of curcumin in mice up to 2.5 fold as compared to curcumin in Tween-20. Furthermore, biodistribution studies of our MNP-CUR formulation demonstrate a significant amount of formulation was able to reach the pancreatic xenograft tumor(s) which suggests its clinical translational potential. In conclusion, our study suggests that our novel MNP-CUR formulation can be valuable for the treatment of pancreatic cancer. Citation Format: Murali M. Yallapu, Mara C. Ebleling, Sheema Khan, Neeraj Chauhan, Brij K. Gupta, Vasudha Sundram, Meena Jaggi, Subhash C. Chauhan. Novel curcumin loaded magnetic nanoparticles for pancreatic cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4503. doi:10.1158/1538-7445.AM2013-4503

Abstract 5500: Ormeloxifene modulates beta-catenin/TCF pathway in colon cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC BACKGROUND: Colorectal cancer is the leading cause of cancer-related death and the third most commonly diagnosed cancer in both males and females in the US. While the overall rates of colon cancer have been steadily falling in the past decade, the rates of colon cancer continue to increase in the under-50 age group. Therefore, developing newer strategies for the prevention and treatment of colon cancer is urgently needed. Aberrant β-catenin signaling has been detected in over 90% of colon cancer and is a central signaling pathway leading to the development of colon cancer. Mutations in components of this pathway lead to the accumulation of β-catenin in the nucleus where it functions as a co-transcription factor to transcribe proto-oncogenes. Therefore, strategies to regulate this specific signaling pathway can be developed as a novel therapeutic modality for the treatment of colon cancer. Ormeloxifene is a synthetic non-steroidal selective estrogen receptor modulator that has been widely used as an oral contraceptive and has demonstrated promising results in the inhibition of breast cancer. Herein, we have investigated the effect of ormeloxifene on colon cancer cell growth and its effect on β-catenin transcriptional activity. MATERIALS AND METHODS: SW480, SW620 colon cancer cells were used to investigate the effect of ormeloxifene (5-20 μM) on various cellular characteristics, including cell proliferation, clonogenic potential, motility and aggregation using standard procedures. β-catenin/T cell factor (TCF) transcription activity was measured by luciferase reporter assays using reporter constructs Topflash or Fopflash and pRL-TK (Renilla luciferase) and with Dual-Glo reagent. The expression and subcellular localization of proteins was determined by immunoblotting and confocal microscopy. RESULTS: Ormeloxifene demonstrated a concentration dependent (5-20 μM) inhibition of cell proliferation in the colon cells. In addition, ormeloxifene very effectively suppressed growth of colon cancer cells in both anchorage dependent and anchorage independent colony formation assays. Furthermore, ormeloxifene effectively inhibited cellular motility of colon cancer cells. Repression of these cellular phenotypes was correlated with attenuation of β-catenin/TCF4 transcriptional activity and the modulation of subcellular localization of β-catenin. Western blot analyses revealed reduction of TCF4 levels upon ormeloxifene treatment. CONCLUSION: Our findings suggest that ormeloxifene effectively inhibits tumorigenic characteristics of colon cancer cells via attenuating the β-catenin/TCF4 signaling pathway. It may therefore be a novel therapeutic modality for the prevention, treatment and management of colon cancer. Citation Format: Vasudha Sundram, Rishi Kumar Gara, Man Mohan Singh, Subhash C. Chauhan, Meena Jaggi. Ormeloxifene modulates beta-catenin/TCF pathway in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5500. doi:10.1158/1538-7445.AM2013-5500