Treatment with cucurbitacin B alone and in combination with gefitinib induces cell cycle inhibition and apoptosis via EGFR and JAK/STAT pathway in human colorectal cancer cell lines

Abstract

<jats:p> The epidermal growth factor receptor (EGFR) associated with signaling pathways, such as Janus kinase (JAK)/signal transducer and activator of transcription (STAT), plays an important role in colorectal cancers (CRCs). Gefitinib (Gef) is an orally active inhibitor targeting the adenosine tri phosphate-binding domain of EGFR, and cucurbitacin B (CuB) is a selective inhibitor of JAK/STAT signaling with potent antitumor activity via suppression of STAT3 phosphorylation, but the underlying mechanism is not clear. We aimed to investigate the apoptotic and antiproliferative effects of CuB as a single agent and in combination with Gef on both HT-29 and HCT-116 cell lines. Cell proliferation, cell cycle distribution, and apoptosis were evaluated using viability assay, fluorescent microscopy, cytotoxicity assay, proliferation, DNA fragmentation, and cleaved caspase 3 levels. Real-time polymerase chain reaction and Western blot analyses were performed to determine the expression of relevant genes and proteins including antiapoptotic, proapoptotic, and cell cycle regulation. EGFR, phosphorylated EGFR (pEGFR), STAT3, and pSTAT3 proteins were evalutaed with Western blot analysis. Our results showed that, compared to CuB alone, CuB plus Gef treatment caused a significant growth and cell cycle inhibition and induced apoptosis in both cell lines. Also CuB plus Gef treatment decreased DNA synthesis rate more effectively than CuB alone. Treatment with CuB alone and in combination with Gef decreased the expression levels of B-Cell CLL/Lymphoma 2 (Bcl-2), BCL2-like 1 (BCL2L1), cyclin D1, pSTAT3, and pEGFR and increased the expression levels of Bcl-2-like protein 4, Bcl-2 homologous antagonist/killer, Bcl-2-associated death promoter, Bcl-2-like protein 11, and p27kip1 levels. Our results suggest that treatment with CuB alone and more likely in combination with Gef may be a considerable alternative therapeutic approach for CRC, at least in vitro. </jats:p>

Comprehensive review of targeted therapy for colorectal cancer

“…Persistent JAK/ STAT activation might also be vital for EGFR-targeted resistance, although the increased level of STAT3 phosphorylation seen in in vivo and in vitro studies was related to gefitinib resistance, which could be overcome by silencing STAT3 in CRC cells. 152,153 Bypass amplification and activation. c-MET and VEGF amplification and activation are discussed in the following parts of this review.…”

Mechanisms of resistance to anti-EGFR therapy in colorectal cancer

“…A similar result was obtained by AS Yar Saglam et al, who demonstrate that combined treatment with cucurbitacin B, a JAK/STAT3 pathway inhibitor, and gefitinib could lead to enhanced antitumor activity in human CRC cells. Therefore, combining EGFR blockade with suppression of JAK/STAT3 signaling is more effective in inhibiting CRC cell growth than inhibition of either pathway alone [73]. …”

MiR-15b is a key regulator of proliferation and apoptosis of chondrocytes from patients with condylar hyperplasia by targeting IGF1, IGF1R and BCL2

“…In addition to the direct regulation of miR-15b on IGF1, IGF1R and BCL2 in CH chondrocytes, whether the indirect regulation exists or not needed to be addressed. JAK/STAT and PI3K/AKT are classical signal pathways for BCL2 and IGF1/IGF1R, individually 24,25 . Hence, whether miR-15b was able to modulate the expression of JAK/STAT and PI3K/AKT in these cells was investigated.…”