The Inhibition Of Pyruvate Kinase M2Could Partly Explain The Mechanism Of Naphthoquinones Induced Death Of Cancer Cells

From2007to2008, our group has reported a serial of naphthoqinones-shikonin and its derivatives were necroptosis inducers that could bypass cancer drug resistance, which provides a new direction to develop anti-cancer drugs. However, the mechanism was unclear, which brought blindness in developing new drugs. This study tries to uncover the target of shikonin in cancer cells, and explore the mechanism of necroptosis caused by shikonin and its derivatives. Vitamin Ks are another serial of naphthoquinones with similar chemical structure of shikonin, and they are discovered to be potential in cancer therapy. This study also tries to find out whether the mechanism of vitamin Ks in killing cancer cells is similar to shikonin, and which homeostasis they break in cancer cells. First, the result of solid-phase experiment showed that shikonin could bind to pyruvate kinase M2(PKM2) in cell lysate, which implies that PKM2would be an important target of shikonin in vivo. Through the enzyme activity assay, we found that shikonin and its derivatives could inhibit the activity of PKM2, and they were much more efficient than the reported small chemical inhibitor of PKM2compound3. Furthermore, we found that shikonin inhibited isozymes of pyruvate kinase in different extent, and PKM2among its isozymes was inhibited the most by shikonin. Since PKM2is specifically expressed in cancer cells, and the last rate-limiting enzyme of glycolysis, the inhibition of PKM2's activity might induce the inhibition of glycolysis. Through quantifying glucose and lactate in culture medium after the treatment of cancer cells with shikonin, we proved that the consumption of glucose and the production of lactate were reduced, which demonstrated that the rate of glycolysis flux was attenuated by shikonin. And we also found that shikonin was efficient in inhibiting glycolysis of both sensitive cancer cell (MCF-7, MCF-7/Neo) and drug-resistant cancer cells (MCF-7/Adr, MCF-7/Bcl-2and MCF-7/Bcl-xL). Glycolysis is an important resource of energy and anabolism blocks in cancer cells. The inhibition of glycolysis might cause lethal damage to cancer cells. We hypothesized that shikonin might cause necroptosis partly by inhibiting PKM2and glycolysis. According to cell viability assay, we found that cancer cells were more sensitive to shikonin-induced necroptosis after the knock-down of PKM2by siRNA, which suggested that PKM2be related to shikonin-induced necroptosis. After the trasfection of PKM1into cancer cells, the activity of pyruvate kinase increased and cells were less sensitive to shikonin, which proved again that PKM2was an important target of shikonin in killing cancer cells. By similar experiments, we found that vitamin K3and K5inhibited PKM2specifically, as well as the rate of glycolysis. And we also discovered that PKM2was related to the toxicity caused by vitamin K3and K5alone or with doxorubicin. In conclusion, this study demonstrated for the first time that these naphthaquinones were specific inhibitors of PKM2, and the inhibition of PKM2was relevant to cancer cells' sensitivity to naphthoquinones, which provides important clues in developing promising anti-cancer drugs.