Anacardic acid inhibits pancreatic cancer cell growth, and potentiates chemotherapeutic effect by Chmp1A - ATM - p53 signaling pathway.

BACKGROUND: Pancreatic cancer is one of the leading causes of cancer related death and its incidence has risen steadily. Although anticancer drugs have been developed based on the new molecular findings, the drugs have produced unsatisfactory results due to toxicity and resistance. Thus, a complementary therapeutic intervention is urgently needed for pancreatic cancer patients. METHODS: The aim of this study was to assess the potential therapeutic effect of Anacardic acid on pancreatic cancer in vitro and elucidate its underlying mechanisms. Human pancreatic cancer cells were treated with Anacardic acid and assessed for the cytotoxic effect using MTT and spheroid formation assays. Using the same methods, the synergy between Anacardic acid and 5-Fluorouracil or Gemcitabine was determined. To elucidate the underlying molecular mechanisms, Western blot analysis and immunocytochemistry were performed on cancer cells treated with Anacardic acid alone or in combination with 5-Fluorouracil or Gemcitabine. Chromatin Modifying Protein 1A (Chmp1A), Ataxia Telangiectasia Mutated (ATM), and p53 were the primary signaling molecules examined. In addition, Chmp1A was silenced with shRNA to examine the necessity of Chmp1A for the anticancer effect of Anacardic acid, 5-Fluorouracil, or Gemcitabine. RESULTS: Anacardic acid induced an anticancer effect in pancreatic cancer cell lines in a dose dependent manner, and increased the cytotoxicity of 5-Fluorouracil or Gemcitabine in MTT cell viability assays. In spheroid formation assays, spheroids formed were smaller in size and in number upon Anacardic acid treatment compared to control. Mechanistically, Anacardic acid exerted its anticancer activity via the activation of Chmp1A, ATM, and p53. Interestingly, 5-Fluorouracil and Gemcitabine also induced an increase in Chmp1A protein level, suggesting that Chmp1A might mediate the cytotoxic action of chemotherapeutics. Silencing experiments indicate that Chmp1A is required for the action of Anacardic acid, but not for 5-Fluorouracil or Gemcitabine. CONCLUSIONS: Our data suggests that Anacardic Acid might be a promising complementary supplement to slow the initiation or progression of pancreatic cancer.

The planar cell-polarity gene stbm regulates cell behaviour and cell fate in vertebrate embryos.

The gene strabismus (stbm)/Van Gogh (Vang) functions in the planar cell-polarity pathway in Drosophila. As the existence of such a pathway in vertebrates has not been firmly established, we investigated the functions and signalling activities encoded by stbm in vertebrate embryos. In regard to cell fate, inhibition of Stbm function in zebrafish embryos leads to reduction of anterior neural markers, whereas gain of function leads to a rise in the levels of these markers. In regard to cell behaviour, both gain-of-function and loss-of-function assays reveal a role for Stbm in mediating cell movements during gastrulation. Mechanistically, Stbm inhibits Wnt-mediated activation of beta-catenin-dependent transcription while promoting phosphorylation of c-Jun- and AP-1-dependent transcription. This complex effect on intracellular signalling pathways probably involves dishevelled (dsh), as Stbm was found to interact with the Dsh protein, and as Dsh is known to function in both planar cell-polarity and beta-catenin pathways in Drosophila.

Chmp 1A is a mediator of the anti-proliferative effects of all-trans retinoic acid in human pancreatic cancer cells.

BACKGROUND: We recently have shown that Charged multivesicular protein/Chromatin modifying protein1A (Chmp1A) functions as a tumor suppressor in human pancreatic tumor cells. Pancreatic cancer has the worst prognosis of all cancers with a dismal 5-year survival rate. Preclinical studies using ATRA for treating human pancreatic cancer suggest this compound might be useful for treatment of pancreatic cancer patients. However, the molecular mechanism by which ATRA inhibits growth of pancreatic cancer cells is not clear. The objective of our study was to investigate whether Chmp1A is involved in ATRA-mediated growth inhibition of human pancreatic tumor cells. RESULTS: We performed microarray studies using HEK 293T cells and discovered that Chmp1A positively regulated Cellular retinol-binding protein 1 (CRBP-1). CRBP-1 is a key regulator of All-trans retinoic acid (ATRA) through ATRA metabolism and nuclear localization. Since our microarray data indicates a potential involvement of Chmp1A in ATRA signaling, we tested this hypothesis by treating pancreatic tumor cells with ATRA in vitro. In the ATRA-responsive cell lines, ATRA significantly increased the protein expression of Chmp1A, CRBP-1, P53 and phospho-P53 at serine 15 and 37 position. We found that knockdown of Chmp1A via shRNA abolished the ATRA-mediated growth inhibition of PanC-1 cells. Also, Chmp1A silencing diminished the increase of Chmp1A, P53 and phospho-P53 protein expression induced by ATRA. In the ATRA non-responsive cells, ATRA did not have any effect on the protein level of Chmp1A and P53. Chmp1A over-expression, however, induced growth inhibition of ATRA non-responsive cells, which was accompanied by an increase of Chmp1A, P53 and phospho-P53. Interestingly, in ATRA responsive cells Chmp1A is localized to the nucleus, which became robust upon ATRA treatment. In the ATRA-non-responsive cells, Chmp1A was mainly translocated to the plasma membrane upon ATRA treatment. CONCLUSION: Collectively our data provides evidence that Chmp1A mediates the growth inhibitory activity of ATRA in human pancreatic cancer cells via regulation of CRBP-1. Our results also suggest that nuclear localization of Chmp1A is important in mediating ATRA signaling.

Chromatin modifying protein 1A (Chmp1A) of the endosomal sorting complex required for transport (ESCRT)-III family activates ataxia telangiectasia mutated (ATM) for PanC-1 cell growth inhibition.

Chromatin modifying protein 1A (Chmp1A) is a member of the Endosormal sorting complex required for transport (ESCRT)-III family whose over-expression induces growth inhibition, chromatin condensation, and p53 phosphorylation. p53 is a substrate for Ataxia telangiectasia mutated (ATM), which can be activated upon chromatin condensation. Thus, we propose that Chmp1A regulates ATM, and the nuclear localization signal (NLS) is required for ATM activation. Our data demonstrated that over-expression of full-length Chmp1A induced an increase in active, phosphorylated ATM in the nucleus, where they co-localized. It also induced an increase in phospho-p53 in the nucleus, and in vitro ATM kinase and p53 reporter activities. The intensity of phospho-p53 closely followed that of ectopically induced full-length Chmp1A, suggesting a tight correlation between Chmp1A over-expression and p53 phosphorylation. On the other hand, Chmp1A depletion (reported to promote cell growth) had minor effects on phospho-ATM and p53 expression compared to control, which had very little expression of these proteins. NLS-deleted cells showed uniform cytoplasmic-Chmp1A expression and acted like shRNA-expressing cells (cell growth promotion and minimal effect on ATM), demonstrating the significance of NLS on ATM activation and growth inhibition. C-deleted Chmp1A, detected in the cytoplasm at the enlarged vesicles, increased phospho-ATM and p53, and inhibited growth; yet it had no effect on in vitro ATM kinase or p53 reporter activities, suggesting that the C-domain is not required for ATM activation. Finally, ATM inactivation considerably reduced Chmp1A mediated growth inhibition and phosphorylation of p53, showing that Chmp1A regulates tumor growth partly through ATM signaling.

Chmp1A functions as a novel tumor suppressor gene in human embryonic kidney and ductal pancreatic tumor cells.

Chmp1A (Chromatin modifying protein 1A/Charged multivesicular protein 1A) is a member of the ESCRT-III (Endosomal Sorting Complex Required for Transport) family that was shown to function in endosome-mediated trafficking via multivesicular body (MVB) formation and sorting. Recent reports suggest that ESCRT complexes are also involved in cell cycle progression and tumor development. Using in vitro and in vivo model systems, we provide evidence that Chmp1A is a novel tumor suppressor, especially in the pancreas. We demonstrated that short hairpin RNA (shRNA) mediated stable silencing of Chmp1A in HEK 293T cells resulted in an increase of anchorage-independent growth in soft agar assay and tumor formation in xenograft assay. To investigate the involvement of Chmp1A in human tumor development we screened human cancer arrays and pancreatic tissue arrays. We discovered that Chmp1A mRNA and protein was reduced and/or altered (protein) in various human pancreatic tumors. To investigate the biological implication of these data, we either overexpressed or silenced Chmp1A in human pancreatic ductal tumor cells (PanC-1) and studied the effect of these manipulations on cell and tumor growth respectively. Stable overexpression of Chmp1A in PanC-1 cells resulted in cell growth inhibition and tumor xenograft inhibition respectively. In contrast, silencing of Chmp1A in PanC-1 cells resulted in the elevation of cell growth in vitro. Mechanistically, overexpression of Chmp1A strongly increased the protein level of p53 and phospho-P53. Taken together, our data indicates that Chmp1A is a novel tumor suppressor, especially in pancreas and that Chmp1A regulates tumor growth potentially through p53 signaling pathway.