Our results showed that Coix could inhibit the proliferation of GBM in vitro. The IC50 was 17.82mg/ml and the maximum inhibition rate was 52.94%. TMZ has much stronger inhibitory ability on glioma, with IC50 of 0.358 mg / ml and the maximum inhibitory rate of 98.77%.Although Coix has weak inhibition ability, it can partially replace TMZ when combined with TMZ. Coix has been reported to be effective in improving patients' symptoms and anticachexia[9]. Therefore, combined medication is of great significance to reduce the dosage of TMZ and reduce the side effects of chemotherapy. The combination of Coix and TMZ in small doses (TMZ 0.1mg/ml, Coix 2mg / ml) showed a synergistic effect on the inhibition of glioma growth. The transcriptome analysis applied on this dosage reveals interesting mechanisms on synergistic effect of TMZ and Coix.
IFN related genes were highlighted in transcriptome analysis. They were up-regulated by TMZ and down-regulated by Coix. The biology process that down-regulated DEGs enriched in between Coix group and control group are mainly IFN related, including processes directly related to IFN and processes indirectly related to IFN. Since IFN plays an important role in the process of virus resistance,virus infectious disease associated pathways and immune system pathways were overrepresented in the Coix group compared to the control. Most of these biology process and pathways were also overrepresented in TMZ group compared to the control, however, enriched with up-regulated DEGs.
Taking further investigation, we found the DEGs enriched in these pathways and biology process were mainly virus recognition and IFN response related. They sense and recognize abnormal genetic materials, such like viral dsRNA, and signaling to erect a line of defense. For example, IFIH1 which was up-regulated 2.7 fold by TMZ encodes a pattern recognition receptor that senses viral dsRNA and activates type I IFN antiviral signaling. IFN receptorJAK3 and signal transducers (STAT1 and STAT2) were also up-regulated. IFN induces an antiviral state of cells, and four groups of genesare induced to form this state of cells. All of them have members up-regulated by TMZ, including OAS1, OAS2 and OAS3 in OAS group, MX1 and MX2 in Mx group, ISG15 in ISG group. Besides IFN signal, the induction of the four group genes were also depend on abnormal genetic materials. PKRs and OASs are synthesized in an inactive form and utilize dsRNA as a cofactor. Mx proteins act by recognizing nucleocapsid-like structures[10].
Why were IFN related pathways and biology process up-regulated by TMZ treatment? According to previous reports we described above, abnormal genetic materials are key affecters of IFN induction and response. The inductions of IFN related genes are depending on them. On the other hand, it is known that the mechanism of TMZ against glioma cells is DNA methylation, leading to gene mismatch accumulation and finally apoptosis[4].TMZ induces DNA damage and the drug resistance was caused by the repairing of the damage. Thus, we suggest that the abnormal genetic materials caused by TMZ treatment induced DNA damage can be sensed by IFN related genes and activates antiviral IFN signaling, causing the up-regulation of IFN related genes and the overrepresented IFN related pathways and biology process in TMZ group compared to the control. This suggestion supported by studies on IFN induction that IFN related genes were up-regulated in cells after radiotherapy, and unrepaired DNA damage could induce the production of type I IFN[11].
Most of IFN related genes that up-regulated by TMZ were down-regulated by Coix. We suppose the down-regulation of these genes act on reverse drug resistance of TMZ. The up-regulation of IFN related genes induced by TMZ treatment can reduce the effect of chemotherapy, which has been proved by relevant studies.[12] Under most situations, the STAT1/IFN pathway transmits a cytotoxic signal either in response to DNA damage or to IFNs. But under the chronic stimulation of DNA damage, it might have selected for the failure to transmit a cytotoxic signal and instead results in pro-survival signals mediated by STAT1 and other IFN-related DNA damage resistance signature genes[13, 14]. Previous studies also found that IFN not only play an important role in the process of virus resistance, but also have a certain impact on tumor proliferation. And IFN related genes are involved in tumor growth. For example, CXCL10 plays a key role in inhibiting glioma growth. Some studies have shown that the expression of CXCL10 is up-regulated in gliomas and increases with the increase of malignant degree of gliomas, and promotes early tumor growth by increasing tumor neovascularization[15–17]. IFIH1 can up-regulate the expression of CXCL10 by activating ISG56 through positive feedback[18]. RSAD2 and MAP2K6 play a key role in p38 MAP kinase signal cascade and may inhibit glioma proliferation by down-regulating p38 MAP pathway[19, 20]. In addition, IL-12 is up-regulated, which has been proved to have anti endogenous brain tumors in previous studies[21, 22]. Thus, the down-regulation of IFN related genes by Coix weakens the pro-survival signals making by IFN related genes, thereby, reverses the drug resistance of TMZ and causes synergistic effect of TMZ combined with Coix.
Besides IFN related genes, genes in cholesterol metabolism pathway were also highlighted in the study. Studies showed that ANGPTL4 promotes the migration, invasion and tubular formation of C6 cells in vitro. The tubular channel formed by it may play a role in the blood supply and metastasis of glioma. And ANGPTL4 can activate ferroptosis by activating NOX2.[23][24]. The fact that genes belong to ferroptosis were indeed regulated in the Coix group in our result supported the previous study. ABCA1 is a member of the ATP binding cassette (ABC) transporter superfamily[25]. Over-expression of ABCA1 can cause the mediated intracellular cholesterol imbalance, which may contribute to the growth and distant metastasis of primary tumors[26]. In the latest study, TMZ outflow was controlled by ABCA1 activity. The expression level of ABCA1 gene was an indicator of TMZ treatment efficiency and participated in the mechanism of TMZ resistance[27]. Therefore, the down-regulation of ABCA1 may be one of the reasons why Coix enhances the efficacy of TMZ.