| Winter worm and summer grass (WWSG) is one of the most valued traditional Chinese medicines for fighting cancer, increasing longevity, and improving immunity. It consists of the entomopathogenic fungus Cordyceps sinensis and its natural lepidopteran host Hepialus armoricanus. Using the water extract of Cordyceps militaris, a sibling species of C. sinensis cultivated on an artificial host the silkworm Bombyx mori pupae, I have found that the C. militaris extract inhibited growth of MCF-7 human breast cancer cells in a dose- and time-dependent manner, with IC50 value at about 11 mug/ml when MCF-7 cells were incubated in the C. militaris extract-containing medium for 96 h. The inhibitory effect of the C. militaris extract on MCF-7 cells was irreversible. Real-time PCR analysis revealed that the C. militaris extract induced the transcriptional expression of early-response pro-apoptotic marker genes Bax and Bim in MCF-7 cells incubated with the C. militaris extract for 6 h or longer and induced the late-response apoptotic genes Apaf-1 and Caspase-7 in MCF-7 cells after 48 h incubation. The transcript of another gene Cytc which encodes cytochrome c, an essential element in mitochondria-mediated apoptosis, was also induced in MCF-7 cells after 48 h incubation with the C. militaris extract. The anti-apoptotic gene Bcl-2 transcript was suppressed in MCF-7 cells after 6 h incubation with the C. militaris extract. All data indicate the C. militaris extract inhibited MCF-7 cells growth by sequentially inducing the expression of proapoptotic and apoptotic genes Bim, Bax, Cytc, Apaf-1 and Caspase-7. It also inhibited the transcriptional expression of the anti-apoptotic gene Bcl-2, a gene encoding Bcl-2 which contributes to the initiation of this apoptosis pathway by activating Bax. The activation of caspases leads to the subsequent DNA fragmentation as detected in TUNEL system. Although the C. militaris extract also prevented MCF-10A normal breast cells from growth, MCF-10A cells quickly recovered to their normal growth rate when removed from the C. militaris extract-containing medium to fresh medium.;In addition to the apoptotic genes, the levels of the methyltransferase gene DNMT1 and DNMT3a transcripts were also suppressed in MCF-7 cells incubated with the C. militaris extract for 6 h. This result indicates that less methylation in some tumor-suppressor genes may potentially lead to regained expression of these genes and subsequent inhibition of cancer cell growth.;In summary, these findings suggest that the C. militaris extract inhibits human breast cancer cell growth through an apoptosis cascade by inducing pro-apoptotic and suppressing anti-apoptotic marker gene expression. Moreover, the C. militaris extract reduced DNA methylation through the suppression of methyltransferase transcripts, leading to the recovery of tumor-suppressor genes and eventually inhibiting tumor cell growth. |
