Such an increase in P21 was accompanied with elevated transcription. Shikonin. A pan-apoptosis inhibitor mainly suppressed Shikonin-induced cell death, suggesting an important part of apoptosis in this process. Intriguingly, Shikonin also triggered autophagy and inhibition of autophagy by depleting essential autophagic genes further improved Shikonin-induced cell death, indicating a protecting part of autophagy. In uncovering the molecular mechanisms underlying these effects of Shikonin, we found that Shikonin induced a powerful upregulation of P21 independent of the P53 status, upregulated autophagy genes, as well as inhibited manifestation of genes required for cell growth. Using mouse tumor models, we confirmed the strong anticancer effect of Shikonin Sieb et Zucc, (Royle) Johnst, or Bunge, is an natural medicine that has been used to treat many kinds of ailments in China and additional Asian and European countries for BMS-3 centuries. A large number of studies have reported a wide range of biological activities of Zicao components including anti-inflammation, anti-oxidative stress, anti-virus, anti-bacteria and anti-cancer in both cultured cells and in animal models SMOC1 (Papageorgiou et al., 1999; Chen et al., 2002; Andujar et al., 2013; Wang et al., 2019). Shikonin is definitely a BMS-3 major component of Zicao and belongs to the naphthoquinone family compound. Consistent with the reported function of Zicao components, Shikonin has shown a broad spectrum of bioactivities including wound healing (Mani et al., 2004), anti-inflammation (Tanaka et al., 1986), anti-HIV (Chen et al., 2003), anti-cancer (Sankawa et al., 1977), and so on. It appears that its toxicity BMS-3 to normal cells and organs is limited; hence, Shikonin has been extensively analyzed as an anti-cancer agent and experienced demonstrated promising effects both and (Papageorgiou et al., 1999; Chen et al., 2002; Andujar et al., 2013; Wang et al., 2019). The molecular mechanisms underlying the anti-cancer activity of Shikonin seemed to be complicated and may depend within the cellular context (Wang et al., 2019). So far, the reported cellular focuses on of Shikonin include the pyruvate kinase isoenzyme M2 (PKM2) (Chen et al., 2011; Lu et al., 2018; Tang et al., 2018b), the MAPK pathway (Mao et al., 2008; Zhao et al., 2015; Shan et al., 2017), HIF1 (Li et al., 2017; Han et al., 2018; Tang et al., 2018b), JNK (Zhai et al., 2017; Lin et al., 2018), PI3K/AKT (Zhang et al., 2015; Zhou et al., 2017; Ni et al., 2018; Tang et al., 2018b), STAT3 (Qiu et al., 2017; Tang et al., 2018a), p16INK4A and p73 (Jang et al., 2015), and PTEN (Nigorikawa et al., 2006; Chen et al., 2018; Zhang et al., 2018). These findings, at one hand, demonstrate that Shikonin can regulate numerous biological processes (Wang et al., 2019). On the other hand, they also illustrate a conundrum as to how precisely Shikonin BMS-3 regulates cellular processes and how such rules contributes to the anticancer activity of Shikonin. In order to understand how Shikonin elicits its anti-cancer activity, in the current study, we systematically investigated the effect of Shikonin on both the short-term proliferation and the long-term survival of various tumor cell lines originated from lung, breast, pancreas, colon and bone and one normal cell collection derived from the liver. We use both chemical and genetic approaches to determine the involvement of cellular processes such as cell cycle, autophagy and apoptosis in the anti-cancer effect of Shikonin. Our data reveal that Shikonin simultaneously induces cell cycle arrest, cell death and autophagy, which collectively control malignancy cell growth, survival and death. Methods Chemicals, Cell Tradition and Reagents Shikonin (>99.0%, #517-89-5) and Z-VAD-FMK (#S7023) were from Selleck Chemicals (Huston, TX, USA). Rapamycin (#D9542) was purchased from Sigma (St. Louis, MO, USA). PEG300 (#P815612) and Tween 80 (#T818928).