Study Of Naturally-occurring Naphthoquinone Analogues Against Cancer Drug Resistance And Metastasis In Vitro And In Vivo

Cancer drug resistance is the major obstacle in chemotherapy.Cellular factors that contribute to drug resistance include drug transporters mediated multidrug resistance and anti-apoptotic factors mediated blocked apoptosis.Currently,there are no clinically available drugs against drug-resistant tumors.In the year 2007,we first reported that shikonin could circumvent cancer drug resistance by induction of necroptosis,a non-apoptotic programmed cell death.It exhibited similar cytotoxic effect against drug-resistant tumor cells and their drug-sensitive parental cells.However,the findings based on a single molecule-shikonin may not have general scientific significance.The reason is straightforward.If only shikonin could induce necroptosis and if a slight chemical modification of shikonin could alter its nature to induce necroptosis,there is no future to develop the drugs based on necroptotic induction.We therefore extended our study from one molecule shikonin to a series of naturally-occurring naphthoquinone analogues,namely,Deoxyshikonin,Acetylshikonin,Isobutyrylshikonin,β,β-Dimethylacrylshikonin, Isovalerylshikonin and(2-Methyl-n-butyl)shikonin,and further investigated if these compounds could also circumvent cancer drug resistance by induction of necroptosis.The MTT assay revealed that all these compounds had similar IC₅₀ values against eight pairs of drug-sensitive and drug-resistant tumor cells in vitro, among which(2-Methyl-n-butyl)shikonin displayed the strongest cytotoxic effect. Necrostatin-1 efficiently inhibited the cell death induced by these analogues,indicating that they induced necroptosis in tumor cells.We further examined if (2-Methyl-n-butyl)shikonin-induced cell death was necroptosis.Cells treated with (2-Methyl-n-butyl)shikonin showed a necrotic morphology such as rupture of cell membrane,vacuolation of cytoplasm and swelling of mitochondria.Autophagosomes were observed in dead cells,indicating the involvement of autophagy in cell death. Nec-1 partially blocked the decrease of mitochondrial membrane potential induced by (2-Methyl-n-butyl)shikonin.Furthermore,no caspase-3,8,9 activation,no obvious hypodiploid peak and no apoptotic DNA ladder were observed in dead cells.The results thus demonstrated that(2-Methyl-n-butyl)shikonin induced necroptosis in tumor cells. The in vivo acute toxicity study revealed that(2-Methyl-n-butyl)shikonin caused a moderate decrease of mice body weight at 100mg/kg dosed intragastrically three times a week.No other obvious toxicity was observed and no mouse died during the experiment. Shikonin administered at the same dosage did not cause any apparent side effect in mice. We further evaluated the antitumor activity of(2-Methyl-n-butyl)shikonin,shikonin and Dox in vivo.The result showed that all three drugs did not obviously inhibit the growth of K562 tumor.On the other hand,(2-Methyl-n-butyl)shikonin,but not shikonin or Dox, displayed a strong inhibitory effect against K562/ADR tumor.We further established a tumor metastasis model in BALB/c mice inoculated with mouse 4T1 breast cancer cells. We found that shikonin significantly inhibited the growth of primary 4T1 tumor,in comparison to controls,although it did not show apparent inhibitory effect on lung metastasis.To sum up,we demonstrated that the naturally-occurring naphthoquinone analogues were a class of necroptotic inducers that could circumvent cancer drug resistance,suggesting their potential applications in clinical cancer therapy.