Gopal Dhar

Crocetin inhibits pancreatic cancer cell proliferation and tumor progression in a xenograft mouse model

Crocetin, a carotenoid compound derived from saffron, has long been used as a traditional ancient medicine against different human diseases including cancer. The aim of the series of experiments was to systematically determine whether crocetin significantly affects pancreatic cancer growth both in vitro and/or in vivo. For the in vitro studies, first, MIA-PaCa-2 cells were treated with crocetin and in these sets of experiments, a proliferation assay using H3-thymidine incorporation and flow cytometric analysis suggested that crocetin inhibited proliferation. Next, cell cycle proteins were investigated. Cdc-2, Cdc-25C, Cyclin-B1, and epidermal growth factor receptor were altered significantly by crocetin. To further confirm the findings of inhibition of proliferation, H3-thymidine incorporation in BxPC-3, Capan-1, and ASPC-1 pancreatic cancer cells was also significantly inhibited by crocetin treatment. For the in vivo studies, MIA-PaCa-2 as highly aggressive cells than other pancreatic cancer cells used in this study were injected into the right hind leg of the athymic nude mice and crocetin was given orally after the development of a palpable tumor. The in vivo results showed significant regression in tumor growth with inhibition of proliferation as determined by proliferating cell nuclear antigen and epidermal growth factor receptor expression in the crocetin-treated animals compared with the controls. Both the in vitro pancreatic cancer cells and in vivo athymic nude mice tumor, apoptosis was significantly stimulated as indicated by Bax/Bcl-2 ratio. This study indicates that crocetin has a significant antitumorigenic effect in both in vitro and in vivo on pancreatic cancer. [Mol Cancer Ther 2009;8(2):315–23]

Breast cancer cells secreted platelet‐derived growth factor‐induced motility of vascular smooth muscle cells is mediated through neuropilin‐1

Motility of vascular smooth muscle cells (SMCs) is an essential step for both normal and pathologic angiogenesis. We report here that breast tumor cells, such as MCF‐7 and MDA‐MB‐231, can modulate this SMC migration. We present evidence that the tumor cell‐derived platelet‐derived growth factor (PDGF) is the key regulator of vascular SMCs motility induced by breast cancer cells. PDGF significantly upregulates neuropilin‐1 (NRP‐1) mRNA expression and protein production in aortic smooth muscle cells (AOSMCs) and depletion of NRP‐1 production by AOSMCs with specific short hairpin RNA (shRNA) prevents the PDGF‐dependent migration of vascular SMCs. Moreover, we demonstrate that PDGF physically interacts with NRP‐1. We propose that tumor‐derived PDGF and NRP‐1 of AOSMCs function as a relay system that promotes motility of vascular SMCs.

CCN5/WISP-2 expression in breast adenocarcinoma is associated with less frequent progression of the disease and suppresses the invasive phenotypes of tumor cells.

Although previous in vitro studies predicted that CCN5/WISP-2 may act as an anti-invasive gene in breast cancer, the distribution pattern of CCN5 in breast cancer samples is conflicting. Thus, we systematically investigated the CCN5 expression profile in noninvasive and invasive breast tumor samples and its functional relevance in breast cancer progression. The studies showed that CCN5 expression is biphasic, such that in normal samples CCN5 expression is undetectable, whereas its expression is markedly increased in noninvasive breast lesions, including atypical ductal hyperplasia and ductal carcinoma in situ. Further, CCN5 mRNA and protein levels are significantly reduced as the cancer progresses from a noninvasive to invasive type. Additionally, we showed that CCN5 mRNA and protein level was almost undetectable in poorly differentiated cancers compared with the moderately or well-differentiated samples and its expression inversely correlated with lymph node positivity. The result was further supported by evaluating the RNA expression profile in microdissected sections using real-time PCR analysis. Therefore, our data suggest a protective function of CCN5 in noninvasive breast tumor cells. This hypothesis was further supported by our in vitro studies illuminating that CCN5 is a negative regulator of migration and invasion of breast cancer cells, and these events could be regulated by CCN5 through the modulation of the expression of genes essential for an invasive front. These include Snail-E-cadherin signaling and matrix metalloproteinase (MMP)-9 and MMP-2. Collectively, these studies suggest that the protective effect of CCN5 in breast cancer progression may have important therapeutic implications.

Gain of Oncogenic Function of p53 Mutants Induces Invasive Phenotypes in Human Breast Cancer Cells by Silencing CCN5/WISP-2

CCN5/WISP-2 is overexpressed in noninvasive breast cancer cells and tissue samples, whereas its expression is minimal or undetected in invasive conditions. CCN5/WISP-2 has been considered as an antiinvasive gene because CCN5/WISP-2 silencing augments the invasive phenotypes in vitro. However, the mechanism of silencing of CCN5 during the progression of the disease has been elusive. Because p53 mutations are associated with breast cancer progression and have been shown to correlate inversely with CCN5/WISP-2 expression in other cancer cell types, the objective of this study was to explore whether p53 mutants suppress CCN5 expression in breast tumor cells resulting in the progression of this disease. We found CCN5 expression is inversely correlated with the mutational activation of p53 in human breast tumor cells. The ectopic expression of p53 mutants in ER-positive noninvasive breast tumor cells silenced the CCN5/WISP-2 expression and enhanced invasive phenotypes, including the induction of morphologic changes from the epithelial-to-mesenchymal type along with the alterations of hallmark proteins of these cell types and an augmentation of the migration of these cells. The suppression of CCN5 by the p53 mutants can be nullified by estrogen signaling in these cells through the transcriptional activation of the CCN5 gene. Moreover, the invasive changes can be imitated by blocking the CCN5/WISP-2 expression through RNA interference or can be reversed by the addition of CCN5/WISP-2 recombinant protein in the culture. Thus, these studies suggest that CCN5 inactivation could be an essential molecular event for p53 mutant-induced invasive phenotypes.

Tumor cell-derived PDGF-B potentiates mouse mesenchymal stem cells-pericytes transition and recruitment through an interaction with NRP-1

BackgroundNew blood vessel formation, or angiogenic switch, is an essential event in the development of solid tumors and their metastatic growth. Tumor blood vessel formation and remodeling is a complex and multi-step processes. The differentiation and recruitment of mural cells including vascular smooth muscle cells and pericytes are essential steps in tumor angiogenesis. However, the role of tumor cells in differentiation and recruitment of mural cells has not yet been fully elucidated. This study focuses on the role of human tumor cells in governing the differentiation of mouse mesenchymal stem cells (MSCs) to pericytes and their recruitment in the tumor angiogenesis process.ResultsWe show that C3H/10T1/2 mouse embryonic mesenchymal stem cells, under the influence of different tumor cell-derived conditioned media, differentiate into mature pericytes. These differentiated pericytes, in turn, are recruited to bind with capillary-like networks formed by endothelial cells on the matrigel under in vitro conditions and recruited to bind with blood vessels on gel-foam under in vivo conditions. The degree of recruitment of pericytes into in vitro neo-angiogenesis is tumor cell phenotype specific. Interestingly, invasive cells recruit less pericytes as compared to non-invasive cells. We identified tumor cell-secreted platelet-derived growth factor-B (PDGF-B) as a crucial factor controlling the differentiation and recruitment processes through an interaction with neuropilin-1 (NRP-1) in mesenchymal stem cells.ConclusionThese new insights into the roles of tumor cell-secreted PDGF-B-NRP-1 signaling in MSCs-fate determination may help to develop new antiangiogenic strategies to prevent the tumor growth and metastasis and result in more effective cancer therapies.

Insulin-like Growth Factor-1 (IGF-1) Induces WISP-2/CCN5 via Multiple Molecular Cross-talks and Is Essential for Mitogenic Switch by IGF-1 Axis in Estrogen Receptor–Positive Breast Tumor Cells

Previously, we have shown that the expression of Wnt-1-induced signaling protein-2 (WISP-2), also known as CCN5, can be regulated by multiple stimulants in estrogen receptor (ER)-positive breast tumor cells to exert their mitogenic action in these cells. Here, we show that insulin-like growth factor-1 (IGF-1), a strong mitogen, enhanced the expression of the WISP-2/CCN5 gene parallel with the induction of proliferation of ER-positive breast tumor cells. An additive effect was also seen in combination with estrogen. Perturbation of IGF-1-induced WISP-2/CCN5 expression by WISP-2-specific RNA interference impaired the mitogenic action of IGF-1 on ER-positive breast tumor cells. Furthermore, the studies have shown that the multiple molecular cross-talks and side-talks among IGF-1R, ER-alpha, and phosphatidylinositol 3-kinase (PI3K)/Akt signaling molecules are required to induce WISP-2/CCN5 mRNA by IGF-1 in ER-positive, noninvasive breast tumor cells. Because a pure anti-ER ICI 182,780 is not only able to suppress the up-regulation of WISP-2/CCN5 mRNA expression by IGF-1, it also suppresses the PI3K/Akt activity induced by IGF-1 in MCF-7 cells; we anticipate that the membrane ER receptor may participate in this event. Collectively, these studies propose for the first time that WISP-2/CCN5 is an integral signaling molecule in mitogenic action of IGF-1 axis in ER-positive human breast tumor cells.

Molecular basis of protective effect by crocetin on survival and liver tissue damage following hemorrhagic shock.

Hemorrhagic shock (HS) causes reduction of cellular energy stores, as measured by levels of ATP and ADP. Furthermore, energy depletion may cause mitochondrial damage, which in turn leads to cell death by apoptosis. The hypothesis of the present study is that by enhancing the recovery of cellular ATP and ADP and mitochondrial damage can be reduced, and the extent of apoptosis minimized. Crocetin, a carotenoid compound, appears to enhance the diffusion of oxygen in aqueous solution, and hence may improve energy stores both to the cell and within it. HS was produced in Sprague–Dawley rats by withdrawing blood from the carotid cannula until a mean arterial pressure of 35–40 mm Hg was reached, and then maintained by further withdrawals of blood for 30 and 60 min. Crocetin was administered 2–4 mg/kg in resuscitation fluid through venus cannula and the animals survived for 24–48 h after HS. Experiments designed to promote tissue reconstitution of ATP using crocetin indicate that these approaches are successful in increasing ATP post-hemorrhage and survival. Crocetin treatment also inhibited cellular damage as indicated by increase of Bcl-2 following decrease in cytosolic cytochrome c and caspase-3 after resuscitation. The prolonged energy deficit seen after hemorrhagic shock can produce late damage and rapid restoration of ATP levels to baseline can reduce apoptosis. In conclusions, crocetin can minimize the cellular damage as evidenced by apoptosis and increased the survival of rats. (Mol Cell Biochem 278: 139–146, 2005)

2‐Methoxyestradiol modulates β‐catenin in prostate cancer cells: A possible mediator of 2‐methoxyestradiol‐induced inhibition of cell growth

2‐Methoxyestradiol (2‐ME2) is a novel anticancer agent because of its ability to potentiate apoptotic cell death and inhibit cancer cell growth and angiogenesis. The modes of action of this agent, however, have not yet been fully elucidated. In our study, we have investigated whether 2‐ME2 is able to modulate β‐catenin signaling in prostate cancer cells, which is one of the major players in cell–cell adhesion, proliferation, apoptosis and carcinogenesis. We found that β‐catenin levels were significantly upregulated by 2‐ME2 in a dose‐dependent manner in androgen dependent and independent prostate cancer total cellular extracts. We further show that β‐catenin levels were significantly increased in the membrane fraction, while nuclear fractions of β‐catenin were downregulated in the 2‐ME2‐treated cells. Accumulation of dephospho‐β‐catenin (nondegraded form) parallel with Bcl‐2 and Cyclin D1 downregulation was also achieved after 2‐ME2 treatment. Moreover, we demonstrate that the β‐catenin production by 2‐ME2 is mediated through the MEK/ERK‐2 signaling pathway. Collectively, these results suggest that the cytostatic effect of 2‐ME2 may be mediated through the prevention of the translocation of β‐catenin to the nucleus parallel with an increase in cell–cell adhesion by increasing membrane β‐catenin production, eventually preventing cell migration. Moreover, dephospho‐β‐catenin accumulation by 2ME2 in the cytoplasm may contribute to the induction of apoptosis of these cells. Finally, studies testing the efficacy of 2‐ME2 in human prostate cancer are warranted to determine whether the inhibition of the expected loss of membranous β‐catenin and the upregulation of nuclear β‐catenin can prevent prostate cancer development and progression.

2-Methoxyestradiol Inhibits Barrett's Esophageal Adenocarcinoma Growth and Differentiation through Differential Regulation of the β-Catenin–E-Cadherin Axis

The purpose of this study was to evaluate whether 2-methoxyestradiol (2-ME2), a promising anticancer agent, modulates Barretts esophageal adenocarcinoma (BEAC) cell growth and behavior through a cellular pathway involving β-catenin in partnership with E-cadherin, which seems to play a critical role in the induction of antitumor responses in cancer cells. We found that 2-ME2 markedly reduced the BEAC cell proliferation through regulating apoptotic machinery such as Bcl-2 and Bax. It may nullify the aggressive behavior of the cells by reducing the migratory behavior. Expressions of β-catenin and E-cadherin and binding of these two proteins is activated in a 2-ME2–dependent fashion in Bic-1 cells. Moreover, overexpressions of these two proteins may be due to the stabilization of these proteins by 2-ME2. We found that 2-ME2–induced antimigratory effects are mediated through the β-catenin–E-cadherin signaling pathways. In view of these results, we determined whether 2-ME2 reduces BEAC tumor growth. Administration of 2-ME2 significantly decreased the growth of BEAC cells xenografted on the flank of nude mice. The evidence presented points out that the effect of 2-ME2 on β-catenin–orchestrated signal transduction plausibly plays a multifaceted functional role to inhibit the proliferation and cell migration of 2-ME2–treated malignant cells and it could be a potential candidate in novel treatment strategies for Barretts esophageal adenocarcinoma. Mol Cancer Ther; 9(3); 523–34

Deficiency of CCN5/WISP-2-Driven Program in breast cancer Promotes Cancer Epithelial cells to mesenchymal stem cells and Breast Cancer growth

Breast cancer progression and relapse is conceivably due to tumor initiating cells (TICs)/cancer stem cells. EMT (epithelial-mesenchymal-transition)-signaling regulates TICs’ turnover. However, the mechanisms associated with this episode are unclear. We show that, in triple-negative-breast cancer (TNBC) cells enriched with TICs, CCN5 significantly blocks cellular growth via apoptosis, reversing EMT-signaling and impairing mammosphere formation, thereby blocking the tumor-forming ability and invasive capacity of these cells. To corroborate these findings, we isolated tumor-initiating side populations (SP) and non-side population (NSP or main population) from MCF-7 cell line, and evaluated the impact of CCN5 on these subpopulations. CCN5 was overexpressed in the NSP but downregulated in the SP. Characteristically, NSP cells are ER-α positive and epithelial type with little tumorigenic potency, while SP cells are very similar to triple-negative ones that do not express ER-α- and Her-2 and are highly tumorigenic in xenograft models. The overexpression of CCN5 in SP results in EMT reversion, ER-α upregulation and delays in tumor growth in xenograft models. We reasoned that CCN5 distinguishes SP and NSP and could reprogram SP to NSP transition, thereby delaying tumor growth in the xenograft model. Collectively, we reveal how CCN5-signaling underlies the driving force to prevent TNBC growth and progression.