In this study, our results revealed for the first time that shikonin promotes time-dependent CypA activation and chromatinolysis, which contributed to the AIF translocation to the nucleus and γ-H2AX formation. In vitro studies have shown that inhibition of CypA with its specific inhibitor CsA, or knockdown not only significantly mitigated shikonin-induced glioma cell death, but also prevented chromatinolysis. The mechanism involves the activated CypA to target the mitochondria, which then triggers the overproduction of mitochondrial superoxide, promotes AIF translocation into the nucleus by depolarizing the mitochondria, and intensifies the formation of γ-H2AX by promoting intracellular accumulation of ROS. Additionally, CypA in the nucleus can form a DNA degradation complex with AIF and γ-H2AX and participate in chromatinolysis. In contrast, inhibition of CypA through its specific inhibitor CsA or knocking it down using siRNA prevented overproduction of mitochondrial superoxide, nuclear translocation of AIF and formation of γ-H2AX, which effectively prevented chromatinolysis induced by shikonin. Furthermore, in the process of shikonin-induced necroptosis of the glioma cell, when specific inhibitors such as Nec-1 and GSK872 are used to inhibit RIP1 and RIP3, or with knockdown of RIP1 and RIP3 using siRNA, the expression of CypA can be inhibited; this proved that RIP1 and RIP3 can regulate CypA. Taken together, our results demonstrate that CypA contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis.
Chromatinolysis has been found to be involved in apoptosis and necrosis[22] [23]. Transmission electron microscopy was considered the “gold standard” for cell death research, and is the most accurate method to distinguish apoptosis and necrosis. Under transmission electron microscope, the apoptotic cells showed clear condensed chromatin, membrane surface curl and apoptotic body formation[24, 25]. Necrosis, which is different from apoptosis in the morphological features, is characterized by the swelling of organelles in the cytoplasm, loss of plasma membrane integrity, and condensation of chromatin into irregular fragments; however, the morphology of nucleus remains intact. In our previous study, the cells treated with shikonin did not appear as clumps of chromatin (a complex of DNA and histone protein) and the nucleus was electron-lucent with an intact nuclear membrane[13]. It is proved that shikonin induced necrosis in the glioma cells. Furthermore, in the process of apoptosis, DNA can be selectively cleaved into fragments of approximately 180-200bp by endonuclease G in cells stressed with apoptosis inducers[17]. However, chromatinolysis was rapid and DNA was cleaved randomly in necrotic cells[26]. This can explain why the nuclear DNA extracted from apoptotic cell showed ladder bands after being electrophoresed agarose gel, while the nuclear DNA extracted from necrotic cells showed a continuous smear band. In this experiment, it was observed that shikonin induced a continuous smear band on the agarose gel and the smear band increased in a time-dependent manner. On the contrary, when using the CypA specific inhibitor CsA or knocking down CypA with siRNA, chromatinolysis and glioma cell death induced by shikonin were both alleviated. It is suggested that CypA can contribute to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis.
Currently, the mechanism of necrotic chromatinolysis is still elusive, but many studies have shown that the nuclear translocation of AIF and the formation of γ-H2AX play an important role in promoting chromatinolysis[21]. As a flavoprotein is normally present in the mitochondrial inter-membrane space, AIF functioned as an endonuclease to degrade DNA after being truncated and redistributes into the nucleus[27]. In our study, after siRNA was used to knockdown AIF, the DNA fragmentation induced by shikonin was observed using agarose gel electrophoresis. Moreover, γ-H2AX can be used as an indicator of DNA DSBs both in vitro and in vivo. When DNA DSBs happens, γ-H2AX can be activated with the activation of ATM and DNAPKcs[9, 10]. In addition, several evidences have shown that CypA is also involved in AIF nuclear translocation and it can positively regulate this process[27, 28]. When AIF translocates into the nucleus, AIF interacts with CypA and phosphorylated H2AX through its C-terminal proline-rich module to form the DNA degradation complex; here, CypA plays the role of an endonuclease resulting in DNA degradation and necroptosis[6, 11, 12]. This may explain why in this study, when using an inhibitor CsA or siRNA to knockdown CypA, expression of AIF, γ-H2AX, p-ATM and other related proteins induced by shikonin was inhibited, and the cell death induced by shikonin was alleviated. In agarose gel electrophoresis, after the use of CsA and siRNA against CypA, the DNA fragmentation induced by shikonin was significantly relieved. In addition, in the neutral comet assay, the increases in the cells with comet tails and the improvement in DNA content in the comet tails induced by shikonin were both inhibited in the presence of CsA. This suggested that CypA participated in the regulation of shikonin-induced DNA DSBs in glioma cells.
Excessive production of ROS by oxidative stress will affect the function of cells. Therefore, inhibiting excessive production of ROS can effectively inhibit the occurrence of oxidative stress-mediated diseases and provide treatment. One of the greatest hazards of oxidative stress is DNA damage, especially DNA DSBs[29]. There is increasing evidence that excessive ROS can lead to nuclear translocation of AIF and formation of γ-H2AX, promoting chromatinolysis. In glioma cells treated with hydrogen peroxide, AIF was found to be released from mitochondria and translocated to the nucleus[14]. Shikonin can induce the overproduction of ROS in cells to cause DNA DSBs, lead to the activation of ATM and DNAPKcs, and promote the formation of γ-H2AX[30]. In this experiment, we found that ROS production induced by shikonin was affected by mitochondrial superoxide. After treatment with the mitochondrial superoxide inhibitor MnTBAP, the ROS content in cells was significantly reduced, the mitochondrial depolarization was significantly alleviated, and the shikonin-induced glioma cell death was inhibited. This indicated that shikonin promoted the death of glioma cells by overproducing mitochondrial superoxide by oxidative stress.
Some studies have shown that CypA can participate in the process of oxidative stress. Cao et al. reported that CypA can increase excessive ROS and promote oxidative stress in cardiomyocytes, and mediate inflammatory response[31, 32]. Moreover, excessive ROS can promote the expression and activation of CypA at the same time[33]. Shikonin can improve the production of ROS in glioma cells in many ways[34]. In this study, we found for the first time that CypA is activated in shikonin-induced necroptosis in a time-dependent manner. The activated CypA can target mitochondrial and cause oxidative stress. The significant increase superoxide in the mitochondria leads to the depolarization of mitochondria and promotes the nuclear translocation of AIF. At the same time, the overproduction of superoxide in mitochondria can increase the content of intracellular ROS and aggravate the formation of γ-H2AX. The application of CsA or MnTBAP can significantly reduce the content of superoxide in mitochondria and intracellular ROS, inhibit nuclear translocation of AIF, and alleviate glioma cell death. This suggests that CypA plays an important role in the oxidative stress induced by shikonin.
Necroptosis can occur in colon cancer, non-small cell lung cancer and breast cancer via activation of RIP1 and RIP3[35–37]. In this study, we confirmed that CypA can participate in the process of shikonin- induced necroptosis, and CypA is the downstream signal of RIP1 and RIP3. The protein expression of p-RIP1, p-RIP3 and CypA increased with the increase in shikonin concentration. CypA expression was inhibited when the specific inhibitors Nec-1 and GSK872 of RIP1 and RIP3 were used or when RIP1 and RIP3 were knocked down by siRNA. It is suggested that RIP1 and RIP3 can regulate the expression of CypA in the process of shikonin-induced necroptosis.
In conclusion, we confirmed for the first time that CypA was involved in shikonin-induced necroptosis. Shikonin induced the activation of CypA in a time-dependent manner. The activated CypA can target mitochondria and trigger the excessive superoxide formation in the mitochondria, which leads to the depolarization of mitochondrial membrane potential, the release of AIF and nuclear translocation of AIF. At the same time, the overproduction of mitochondrial superoxide can increase intracellular ROS and aggravate the formation of γ-H2AX. CypA can also form a DNA degradation complex with AIF and γ-H2AX, which can cause chromatinolysis and promote glioma cell necroptosis (Fig. 8).