Mesobuthus eupeus venom modulates colorectal carcinoma signaling pathways and induces apoptosis

Abstract

Colorectal cancer (CRC) is a significant global health concern, often challenging to treat effectively with conventional methods and burdened by adverse effects. Scorpion venoms offer a unique avenue for exploration, given their ability to disrupt the cell cycle, inhibit growth, and trigger apoptosis. This study delves into the impact of Mesobuthus eupeus (M. eupeus) scorpion venom on the proliferation and progression of colorectal cancer at the molecular level. The total protein concentration in the venom (607.5 µg/mL) also emphasized the rich composition and potential for therapeutic applications. The study reveals that M. eupeus venom effectively reduced the proliferation of DLD-1 and HT-29 colorectal cancer cells in a dose-dependent manner with IC₅₀ values of 4.32 and 7.61 µg/mL, respectively. The venom also impedes cell migration, diminishes colony formation, and triggers apoptosis in the cancer cells. The venom also induced early and late apoptosis in the two cancer cell lines. The human colorectal cancer and apoptotic pathways were clarified at the molecular level using pathway panels, which revealed that 16 genes involved in colorectal cancer increased while 23 decreased. In the HT-29 cell line, 57 genes increased, and 1 decreased following venom treatment. Besides, the mRNA expression of 19 genes involved in the apoptotic pathway was increased, while 22 were reduced in DLD-1 cells. This study underscores the potential of M. eupeus venom as a natural therapeutic approach in the quest for cancer treatments.

Keywords: Mesobuthus eupeus; Colorectal cancer; Cytotoxicity; Molecular mechanism; Venom.

Fig. 1

a The appearance of M. eupeus species scorpion. b The sting and venom at the tip of the stinger of M. eupeus. c The electrophoretic profile of venoms was analyzed on polyacrylamide gel in the presence of SDS under reducing conditions. Lane 1: molecular mass markers, Lane 2–6 different concentrations of venom ranging 5–30 µg. d UV–visible spectrum of M. eupeus venom and analysis and of protein quantity of M. eupeus venom using the BCA method. e Separation by HPLC of M. eupeus venom. The soluble portion of scorpion venom (1 mg of protein) was subjected to chromatographic separation using a reverse-phase C-18 column. The separation was achieved by applying a linear gradient of solvent A (0.1% trifluoroacetic acid in dH₂O) to solvent B (0.10% TFA in acetonitrile), reaching 60% solvent B over a 60-min period

Fig. 2

Cytotoxicity, colony formation, and wound healing effects of M. eupeus venom on cell lines. a Sigmoidal graph of cell viability against log concentration of scorpion venom. b IC₅₀ values (µg/mL) of HT-29 and DLD-1 cell lines. c Colony images of DLD-1 and HT-29 cell lines. d Graph of colony count comparing M. eupeus venom with the control group. e and g Images of in vitro wound healing experiment at 0, 24, and 48 h. f, h Graph of cell count at 0, 24, and 48 h comparing M. eupeus venom with the control group. Results are presented as the mean ± SDV of three independent experiments. *p < 0.01, **p < 0.001, and ***p < 0.0001

Fig. 3

Effects of M. eupeus venom on apoptosis in DLD-1 and HT-29 cell lines. a-1 and b-1 Flow cytometry images of cells in the control group (Non-treated). a-2 and b-2 Flow cytometry images of cells treated with M. eupeus venom (Q1: necrotic cells; Q2: late apoptotic cells; Q3: live cells; Q4: early apoptotic cells). a-3 and b-3 Ratio of necrosis, early apoptosis, and late apoptosis compared to the control group for M. eupeus venom. Results are presented as the mean ± SDV of three independent experiments. **p < 0.001, and ***p < 0.0001

Fig. 4

Effects of M. eupeus venom on genes and proteins involved in apoptosis. a, b Alteration in expression levels of 96 genes involved in the apoptosis panel following the treatment with M. eupeus scorpion venom. c, d Alteration in expression levels of genes BcL-2, Bax, caspase-3, caspase-9, and caspase-12, which play an important role in apoptosis, in DLD-1 and HT-29 cell lines compared to the control group. e, f Representative Immunoblot of proteins Bax, BcL-2, and GAPDH. g, h Analysis of band density of protein expression of Bax and BcL-2 compared to the control group relative to the reference protein (GAPDH). Results are presented as the mean ± SDV of three independent experiments. *p < 0.01, **p < 0.001, and ***p < 0.0001

Fig. 5

Effects of M. eupeus venom on genes and proteins involved in human colorectal carcinoma progression. a, b Alteration in mRNA expression levels of 96 genes involved in the development of colon cancer following the treatment of M. eupeus venom. c, d Alteration in expression levels of genes NF-κB, TP53, SMAD4, APC, BRAF, KRAS, and MLH-1, which play a role in the human colorectal carcinoma progression, in DLD-1 and HT-29 cell lines compared to the control group. e, g Representative Immunoblot of NF-κB, P53, and GAPDH proteins. f, h Analysis of band density of protein expression of NF-κB and P53 compared to the control group relative to the reference protein (GAPDH). Results are presented as the mean ± SDV of three independent experiments. *p < 0.01, and ***p < 0.0001