Effect of pH on antitumor activity of Chinese cobra (Naja atra) cytotoxin-XII

Cytotoxins (CTXs), proteins found in cobra venom, selectively inhibit tumor cell proliferation. Herein, we selected CTX-XII because of its potent antitumor activity to investigate the effect of solution pH on its response. MTT assay results showed significantly higher inhibition rates for CTX-XII at pH 5.72 (75.79 ± 3.48%) than that at pH 7.32 (50.75 ± 3.8%). Flow cytometry demonstrated that apoptosis rates in B16F10 cells induced by CTX-XII were also higher at pH 5.72 (44.92 ± 7.94%) and 4.12 (42.87 ± 1.89%) than at pH 7.32 (23.5 ± 4.02%). Confocal laser scanning microscopy images showed that red fluorescence, representing CTX-XII concentration, was more intense around tumor cells at pH 5.72, with higher levels in the cytoplasm, than at pH 7.32. In the murine melanoma model, tumor weight in the pH 5.72 CTX-XII group (0.45 ± 0.19 g) was significantly lower than that in the pH 7.32 CTX-XII group (0.84 ± 0.42 g). These results indicate that pH has a strong influence on the antitumor activity of CTX-XII, likely due to pH-dependent ionization changes in CTX-XII that increase its affinity for and penetration into tumor cell membranes. This study provides new insights into the antitumor effects of CTXs and factors influencing their activity.