Upregulation of RASSF1A in Colon Cancer by Suppression of Angiogenesis Signaling and Akt Activation.

BACKGROUND/AIMS: Silencing of tumor suppressor genes (TSGs) and promotion of angiogenesis are associated with tumor development and metastasis. However, little is known if angiogenic molecules directly control TSGs and vice versa. METHODS: A regulatory link between angiogenesis and down regulation of TSGs was evaluated using an anti-cancer agent, andrographolide (AGP) in cancer cells, mouse xenograft tissues and patient derived organoids through gene/protein expression, gene silencing, and immunohistochemical analyses. RESULTS: AGP treatment demonstrated significant expression of RASSF1A and PTEN TSGs in colon cancer and other cancer cells, mouse tissues and organoids. Depletion of RASSF1A with siRNA limited cyclin D1 and BAX expression. SiRNA depletion of PTEN, upstream regulator of RASSF1A resulted in a 50% reduction in RASSF1A expression. Histopathological analysis of the AGP treated tumor sections showed significant reduction in vessel size, microvascular density and tumor mitotic index suggesting suppression of angiogenesis. This was corroborated by protein analysis demonstrating significant reductions in angiogenesis signaling pathway molecules VEGF165, FOXM1, and pAkt, but significant elevation of the endogenous angiogenesis inhibitor Tsp-2. Treatment of cells with exogenous VEGF prevented the suppression of angiogenesis signaling by AGP, resulting in sustained expression of pAkt, an upstream down-regulator of RASSF1A. RASSF1A expression remained low in VEGF treated cells despite the addition of AGP. CONCLUSION: Our results demonstrate for the first time that AGP induces RASSF1A expression in colon cancer cells and is dependent on angiogenesis signaling events. Therefore, our research may facilitate novel therapeutic options for advanced colon cancer therapy.

Synergistic potential of dual andrographolide and melatonin targeting of metastatic colon cancer cells: Using the Chou-Talalay combination index method.

Colorectal cancer (CRC) mortality has diminished for decades due to new and improved treatment profiles. However, CRC still ranks as the third most diagnosed cancer in the US. Therefore, a new therapeutic approach is needed to overcome colospheroids inhibition and drug resistance. It is well documented that andrographolide (AGP) and melatonin (MLT) have anti-carcinogenic properties. Our goal was to evaluate their synergistic effects on metastatic colon cancer cells (mCRC) and colospheroids. HT-29 and HCT-15 mCRC cells were simultaneously treated with serial dilutions of AGP and MLT for 24, 48 and 72 h. Cell viability was monitored using the MTT assay. The Chou-Talalay method for drug combination is based on the median effect equation, providing a theoretical basis for the combination index and the isobologram equation. This allows quantitative determination of drug interactions using the CompuSyn software, where CI < 1, = 1, and >1 indicates synergistic, additive, and antagonistic effects respectively. Our results demonstrate that AGP and MLT in combination show synergism with CI values of 0.35293 and 0.34152 for HT-29 and HCT-15 respectively and a fractional inhibition of Fa = 0.50-0.90, as shown by the Fa-CI plot and isobologram. The synergism value was validated in colospheroids (HT-29-s and HCT-15-s) based on morphology, viability, and colony formation and in 5-FU drug resistant cell (HT-29R and HCT-116R) viability. The mechanism(s) of decreased cell viability are due to the induction of ER stress proteins and angiogenic inhibition. Our results provide rationale for using AGP in combination with MLT on mCRC.

Identification of Cross Talk between FoxM1 and RASSF1A as a Therapeutic Target of Colon Cancer.

Metastatic colorectal cancer (mCRC) is characterized by the expression of cellular oncogenes, the loss of tumor suppressor gene function. Therefore, identifying integrated signaling between onco-suppressor genes may facilitate the development of effective therapy for mCRC. To investigate these pathways we utilized cell lines and patient derived organoid models for analysis of gene/protein expression, gene silencing, overexpression, and immunohistochemical analyses. An inverse relationship in expression of oncogenic FoxM1 and tumor suppressor RASSF1A was observed in various stages of CRC. This inverse correlation was also observed in mCRC cells lines (T84, Colo 205) treated with Akt inhibitor. Inhibition of FoxM1 expression in mCRC cells as well as in our ex vivo model resulted in increased RASSF1A expression. Reduced levels of RASSF1A expression were found in normal cells (RWPE-1, HBEpc, MCF10A, EC) stimulated with exogenous VEGF165. Downregulation of FoxM1 also coincided with increased YAP phosphorylation, indicative of tumor suppression. Conversely, downregulation of RASSF1A coincided with FoxM1 overexpression. These studies have identified for the first time an integrated signaling pathway between FoxM1 and RASSF1A in mCRC progression, which may facilitate the development of novel therapeutic options for advanced colon cancer therapy.

Increased reactive oxygen species levels cause ER stress and cytotoxicity in andrographolide treated colon cancer cells.

Chemotherapy continues to play an essential role in the management of many cancers including colon cancer, the third leading cause of death due to cancer in the United States. Many naturally occurring plant compounds have been demonstrated to possess anti-cancer cell activity and have the potential to supplement existing chemotherapy strategies. The plant metabolite andrographolide induces cell death in cancer cells and apoptosis is dependent upon the induction of endoplasmic reticulum stress (ER stress) leading to the unfolded protein response (UPR). The goal of the present study was to determine the mechanism by which andrographolide induces ER stress and to further evaluate its role in promoting cell death pathways. The T84 and COLO 205 cancer cell lines were used to demonstrate that andrographolide induces increased ROS levels, corresponding anti-oxidant response molecules, and reduced mitochondrial membrane potential. No increases in ROS levels were detected in control colon fibroblast cells. Andrographolide-induced cell death, UPR signaling, and CHOP, Bax, and caspase 3 apoptosis elements were all inhibited in the presence of the ROS scavenger NAC. Additionally, andrographolide-induced suppression of cyclins B1 and D1 were also reversed in the presence of NAC. Finally, Akt phosphorylation and phospho-mTOR levels that are normally suppressed by andrographolide were also expressed at normal levels in the absence of ROS. These data demonstrate that andrographolide induces ER stress leading to apoptosis through the induction of ROS and that elevated ROS also play an important role in down-regulating cell cycle progression and cell survival pathways as well.