GMI, an Immunomodulatory Protein from Ganoderma microsporum, Potentiates Cisplatin-Induced Apoptosis via Autophagy in Lung Cancer Cells

Abstract

Cisplatin-based therapy is common in the treatment of several types of cancers, including lung cancers. In our previous study, GMI, an immunomodulatory protein cloned from Ganoderma microsporum, induced a cytotoxic effect in lung cancer cells via autophagy. The aim of this study is to examine the role of GMI in enhancing cisplatin-mediated cell death. On the basis of MTT assay and Combination Index, GMI and cisplatin cotreatment induced a synergistic cytotoxic effect. GMI and cisplatin-induced apoptosis was determined by sub-G1, nuclear condensation, and annexin-V/propidium iodide analyses. On Western blot, expressions of γH2AX and cleaved forms of PARP, caspase-3, and caspase-7 were induced by combined treatment. Akt/mTOR pathway activity, LC3-II expression, and acidic vesicular organelle development demonstrated that cisplatin does not abolish GMI-mediated autophagy. Cyto-ID Green/hoechst 33342 double staining and time-dependent experiment indicated that GMI and cisplatin-treated A549 cells simultaneously express autophagosomes and apoptotic nuclei. To elucidate the role of autophagy in inducing apoptosis by GMI and cisplatin, chemical inhibitors and LC3 shRNA were used to inhibit autophagy. The results showed that 3-methyladenine decreases, while chloroquine increases GMI and cisplatin cotreatment-induced cleavage of caspase-7 and PARP. LC3 silencing abolished activation of apoptosis in A549 cells. Caspase inhibitors and caspase-7 silencing mitigated GMI and cisplatin-elicited cell viability inhibition and apoptosis. This is the first study to reveal the novel function of GMI in potentiating cisplatin-mediated apoptosis. GMI and cisplatin induce apoptosis via autophagy/caspase-7-dependent and survivin- and ERCC1-independent pathway. GMI may be a potential cisplatin adjuvant against lung cancer.

Ganoderma microsporum immunomodulatory protein induces apoptosis and potentiates mitomycin C‐induced apoptosis in urinary bladder urothelial carcinoma cells

“…It is worth noting that the highest expressions of p21 proteins in both GMIP concentration treatments all occurred earlier than caspase activation, indicating the sequential activation in the GMIP pathway from p21 to caspases. It is reported that GMIP induced lung cancer cells go through autophagic cell death . However, in UC cells, the expression level of the regulator protein of switching from autophagy to apoptosis, cleaved Beclin‐1 (50, 37, 35 kDa) were not shown after GMIP treatment (Figure C, 1st row).…”

“…It is reported that GMIP induced lung cancer cells go through autophagic cell death. 19 However, in UC cells, the expression level of the regulator protein of switching from autophagy to apoptosis, cleaved Beclin-1 (50, 37, 35 kDa) were not shown after GMIP treatment ( Figure 3C, 1st row). Moreover, autophagy marker LC3B-II did not show a remarkable increase after GMIP treatment ( Figure 3C, 2nd row), so we suggested the cell death caused by GMIP was mainly through programmed apoptosis rather than autophagy.…”

“…In the null mice model with a xenografted tumour of A549 cells, GMIP treatment has been shown to induce autophagy and reduce tumor growth . Additionally, combined with cisplatin, the treatment could lead to autophagic adaptation and further apoptotic cell death and greatly enhance the efficacy of cisplatin . Moreover, cisplatin‐resistant lung cancer cell lines could also be sensitized to autophagy and advance to apoptosis under combined treatments .…”

“…18 Additionally, combined with cisplatin, the treatment could lead to autophagic adaptation and further apoptotic cell death and greatly enhance the efficacy of cisplatin. 19 Moreover, cisplatin-resistant lung cancer cell lines could also be sensitized to autophagy and advance to apoptosis under combined treatments. 17 Nevertheless, to the best of our knowledge, there has been no clear evidences indicating whether GMIP could solitarily induce apoptosis on cancer cell line or not.…”

Apoptosis induction of colorectal cancer cells HTL-9 in vitro by the transformed products of soybean isoflavones by Ganoderma lucidum

“…Increasing lines of evidences have showed that both G. lucidum (Tang et al, 2006;Loganathan et al, 2014;Hsin et al, 2015) and soybean isoflavones (Kurahashi et al, 2007;Li et al, 2012b) have antitumor activity. In this study, for the first time, the transformation of soybean isoflavones was performed using the homogenate of G. lucidum, and the genistein contents could reach (703.21±4.35) mg/g, with transformed rates of 96.63% and 87.82% of daidzein and genistein, respectively.…”