br Fig iTRAQ based proteomics
Fig. 2. iTRAQ-based proteomics analysis screening of the differentially expressed genes (DEGs) in AIF or vehicle-treated CRC cells. A. Unsupervised clustering of CRC cells based on the expression pattern of DEGs. B. The functional annotation and classification of the DEGs in AIF or vehicle-treated CRC cells. C. Identification of the cellular functions of DEGs.
Fig. 3. RAD51 was elevated in CRC tissues and associated with clinico-pathological features. A. RAD51 mRNA was up-regulated in CRC tissues compared to matched non-cancerous tissues. (B–D). RAD51 expression was positively correlated with lymph node metastasis, TNM stage, and poor outcomes.
were more sensitive to AIF-mediated apoptosis compared to the HCT-116 cells. In addition, AIF also upregulated the levels of the pro-apoptotic cleaved caspase-3 and Bax, and downregulated the anti-apoptotic pro-caspase-3 and Bcl-2 proteins (Fig. 1E). Taken together, AIF suppressed proliferation and induced apoptosis in CRC cells.
3.2. AIF alters the transcriptome of CRC cells
To further explore the underlying mechanisms of the anti -pro-liferative and pro -apoptotic effects of AIF on CRC cells, the tran-scriptomes of the control and AIF-treated cells were sequenced and the differentially expressed genes (DEGs) were identified. The mRNA expression profiles of the cells are summarized in Fig. 2A, which show that 49 genes were up-regulated and 33 genes were down-regulated in the AIF-treated relative to control cells. The DEGs were then functionally annotated and classified into the biological pro-cess, cell component and molecular function groups, which indicated that the most significant DEGs were implicated in cell division and DNA replication ( Fig. 2B and C). Therefore, AIF likely exerts its ef-fects in CRC cells by arresting Rottlerin and thus altering prolifera-tion rates.
3.3. RAD51 was up-regulated in CRC tissues and associated with poor prognosis in CRC patients
Microarray analysis showed significantly higher abundance of RAD51 in CRC tissues compared to the matched non-cancerous tissues. We validated the microarray results in a relatively large cohort (n = 47) by western blot. As shown in Fig. 3A, RAD1 protein expression levels
were ~2.5-fold higher in the CRC tissues compared to the matched healthy tissues. In addition, a positive correlation was observed be-tween RAD51 levels and lymph node metastasis and TNM stage, in-dicating that RAD51 functions as an oncogene in CRC (Fig. 3B–C, Table 1). Furthermore, high RAD51 levels in CRC tissues were asso-ciated with poor overall survival of the patients (Fig. 3D). Taken to-gether, RAD51 is a potential prognostic indicator as well as a ther-apeutic target for CRC.
3.4. AIF induces DNA-DSBs in CRC cells by down-regulating RAD51
To summarize the results above, CRC tissues had significantly higher RAD1 expression levels, and AIF significantly downregulated RAD51 mRNA in CRC cells. We hypothesized therefore that the in-hibitory effect of AIF on CRC cell survival was mediated via RAD1 down-regulation. In support of our hypothesis, both 5 and 10 μM AIF effectively decreased the level of RAD51 protein in HCT-116 and SW480 cells (Fig. 4A). Given that RAD51 is involved in DNA DSB re-pair, we next speculated that AIF blocks RAD51-mediated DNA breakage repair in the CRC cells. DSB formation is characterized by the phosphorylation of H2AX at ser-139 to γ-H2XA, which serves as a re-liable marker of DNA damage . As shown in Fig. 4B, few γ-H2AX foci were observed in the vehicle-treated cells, and was dramatically increased upon AIF treatment. In addition, 5 and 10 μM AIF increased the levels of phosphorylated H2AX by 1.8 and 2.8-folds respectively compared to that in the control cells (Fig. 4C). Taken together, AIF increases DNA DSBs by inhibiting DNA repair via RAD51 down-regulation.
Fig. 4. AIF downregulated RAD51 and upregulated γH2AX in CRC cell lines. (A, C). Western blots showing relative RAD51 and γH2AX levels in AIF-treated CRC cells, normalized to GAPDH. B. Number of γH2AX foci in the AIF-treated and control cells. Data are presented as mean ± SD from three independent experiments, **p < 0.01.
3.5. RAD51 silencing augmented the effects of AIF in CRC cells
To further investigate whether RAD51 contributed to the anti-cancer effect of AIF against CRC cells, we silenced RAD51 expression using specific siRNA (Fig. 5A). In line with the results so far, AIF (10 μM) treatment decreased RAD51 protein levels in CRC cells (Fig. 5A). Furthermore, RAD51 knockdown significantly decreased γ-H2AX levels when compared with to the control siRNA-transfected cells, which was partly alleviated by AIF treatment (Fig. 5B). In addi-tion, RAD1 knockdown also enhanced the anti-proliferative effect (Fig. 5C), as well as the apoptosis-inducing effects of AIF (Fig. 5D) in the HCT-116 and SW480 cells. Inhibition of RAD1 increased the per-centage of apoptotic cells in the AIF-treated HCT-116 and SW480 cell lines from 21% and 28% to 30% and 44% respectively (Fig. 5D). Consistent with this, knockdown of RAD51 further augmented the ef-fects of AIF on the expression levels of apoptosis-related proteins (Fig. 5E). Taken together, RAD51 is an important mediator of AIF-regulated cell growth and apoptosis in CRC.