Proteins from Lignosus tigris with selective apoptotic cytotoxicity towards MCF7 cell line and suppresses MCF7-xenograft tumor growth

Abstract

Background Lignosus tigris, a recently discovered species of the unique Lignosus family, has been traditionally used by the indigenous communities in Peninsular Malaysia to treat various ailments and as an alternative medicine for cancer treatment. The L. tigris cultivar sclerotia (Ligno TG-K) was found to contain numerous bioactive compounds with beneficial biomedicinal properties and the sclerotial extract exhibited potent antioxidant activity. However, the anticancer property of the Ligno TG-K including in vitro and in vivo antitumor effects as well as its anticancer active compounds and the mechanisms has yet to be investigated. Methods The cytotoxicity of the Ligno TG-K against human breast (MCF7), prostate (PC3) and lung (A549) adenocarcinoma cell lines was evaluated using MTT cytotoxicity assay. The cytotoxic mechanisms of the active high molecular weight proteins (HMWp) fraction were investigated through detection of caspases activity and apoptotic-related proteins expression by Western blotting. The in vivo antitumor activity of the isolated HMWp was examined using MCF7 mouse xenograft model. Shotgun LC-MS/MS analysis was performed to identify the proteins in the HMWp. Results and Discussion Cold water extract of the sclerotia inhibited proliferation of MCF7, A549 and PC3 cells with IC50 ranged from 28.9 to 95.0 µg/mL. Bioassay guided fractionation of the extract revealed that HMWp exhibited selective cytotoxicity against MCF7 cells via induction of cellular apoptosis by the activation of extrinsic and intrinsic signaling pathways. HMWp activated expression of caspase-8 and -9 enzymes, and pro-apoptotic Bax protein whilst inhibiting expression of tumor survivor protein, Bcl-2. HMWp induced tumor-cell apoptosis and suppressed growth of tumor in MCF-7 xenograft mice. Lectins, serine proteases, RNase Gf29 and a 230NA deoxyribonuclease are the major cytotoxic proteins that accounted for 55.93% of the HMWp. Conclusion The findings from this study provided scientific evidences to support the traditional use of the L. tigris sclerotia for treatment of breast cancer. Several cytotoxic proteins with high abundance have been identified in the HMWp of the sclerotial extract and these proteins have potential to be developed into new anticancer agents or as adjunct cancer therapy.

Antioxidant and Cytotoxic Effects and Identification of Ophiocordyceps sinensis Bioactive Proteins Using Shotgun Proteomic Analysis

“…The separated protein bands were excised into 10 gel sections, where the gel sections were destained, reduced with dithiothreitol, alkylated with iodoacetamide and tryptic digested with trypsin protease (Thermo Scientific TM , Pierce™, Rockford, IL, USA) ( 21 ). Analysis was performed using an Agilent 1260 HPLC-Chip/MS Interface, coupled with Agilent 6550 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA), following the protocol as described previously ( 21 , 22 ). The National Center for Biotechnology Information (NCBI) database of Ophiocordycipitaceae (non-redundant) was used for mass spectra searches that were performed using the Agilent Spectrum Mill MS Proteomics Workbench software packages.…”

Review of Malaysian Medicinal Plants with Potential Anticancer Activity

“…Then, the study concluded that Kava constituents produced a promising outcome for the treatment of oral carcinogenesis and a translation into in vivo studies. The next plant is Lignosus tigris from also known as Cendawan susu rimau consists of cytotoxic protein including cultivar sclerotia (Ligno TF-K), lectins, a serine protease, RNase Gf29, and 230NA deoxyribonuclease (Kong et al, 2020). From all the compounds isolated from Lignosus tigris, Ligno TF-K (a high molecular weight (HMW) protein) is the most abandon cytotoxic protein isolated using cold water and also showed a stronger cytotoxic effect with IC 50 value at 4.23 µg/mL against MCF7 breast cancer cells throughout in vitro study (Kong et al, 2020).…”

“…The next plant is Lignosus tigris from also known as Cendawan susu rimau consists of cytotoxic protein including cultivar sclerotia (Ligno TF-K), lectins, a serine protease, RNase Gf29, and 230NA deoxyribonuclease (Kong et al, 2020). From all the compounds isolated from Lignosus tigris, Ligno TF-K (a high molecular weight (HMW) protein) is the most abandon cytotoxic protein isolated using cold water and also showed a stronger cytotoxic effect with IC 50 value at 4.23 µg/mL against MCF7 breast cancer cells throughout in vitro study (Kong et al, 2020). Besides that, in further exploring the mechanisms of cytotoxic of this HMW protein, the 100% saturated ammonium sulfate was used and tested on the MCF7 cell line and found that it effectively inhibited the proliferation of MCF7 cell and was selective with IC 50 value at 0.76±0.19 µg/mL which was more potent than extract using cold water (Kong et al, 2020).…”

“…From all the compounds isolated from Lignosus tigris, Ligno TF-K (a high molecular weight (HMW) protein) is the most abandon cytotoxic protein isolated using cold water and also showed a stronger cytotoxic effect with IC 50 value at 4.23 µg/mL against MCF7 breast cancer cells throughout in vitro study (Kong et al, 2020). Besides that, in further exploring the mechanisms of cytotoxic of this HMW protein, the 100% saturated ammonium sulfate was used and tested on the MCF7 cell line and found that it effectively inhibited the proliferation of MCF7 cell and was selective with IC 50 value at 0.76±0.19 µg/mL which was more potent than extract using cold water (Kong et al, 2020). The mechanisms of HMW protein showed through in vivo study on Athymic NCR nude mice that it can induce apoptosis where it was hypothesized to be mediated by caspase activation which induces caspase-8 and -9 via the extrinsic and intrinsic apoptotic pathways.…”

“…At the same time, caspase-3 was absent in the cancerous cell. Both of these pathways will eventually activate by caspase-7, resulting in subsequent cell death (Kong et al, 2020). Following that, another novel, the first-time-studied protein was discovered in mushrooms, specifically in Cendawan susu rimau (Kong et al, 2020).…”