C1QBP regulates apoptosis of renal cell carcinoma via modulating xanthine dehydrogenase (XDH) mediated ROS generation

Abstract

Background: Complement component 1 Q subcomponent binding protein (C1QBP) plays a vital role in the progression and metabolism of cancer. Studies have shown that xanthine dehydrogenase (XDH)-derived reactive oxygen species (ROS) accelerates tumor growth, and also induces mutations or produces cytotoxic effects concurrently. However, the role of C1QBP in metabolism, oxidative stress, and apoptosis of renal cell carcinoma (RCC) cells have not yet been explored. Methods: Metabolomics assay was applied to investigate the role of C1QBP in RCC metabolism. C1QBP knockdown and overexpression cells were established via lentiviral infection and subjected to apoptosis and ROS assay in vitro. RNA stability assay was applied to characterize the mechanism of C1QBP regulating XDH transcription. In vivo, orthotopic tumor xenografts assay was performed to investigate the role of C1QBP in RCC progression. Results: Metabolomics investigation revealed that C1QBP dramatically diminished the hypoxanthine content in RCC cells. C1QBP promoted the mRNA and protein expression of hypoxanthine catabolic enzyme XDH. Meanwhile, C1QBP may affect XDH transcription by regulating the mRNA level of XDH transcriptional stimulators IL-6, TNF-α, and IFN-γ. Moreover, the expression of C1QBP and XDH was lower in RCC tumors compared with the tumor-associated normal tissues, and their down-regulation was associated with higher Fuhrman grade. C1QBP significantly increased ROS level, apoptosis, and the expression of apoptotic proteins such as cleaved caspase-3 and bax/bcl2 via regulating XDH. Conclusion: C1QBP promotes the catabolism of hypoxanthine and elevates the apoptosis of RCC cells by modulating XDH-mediated ROS generation.

Keywords: Apoptosis; C1QBP; ROS; Renal cell carcinoma; XDH.

Figure 1

C1QBP regulates hypoxanthine level in RCC. ACHN and 786-O cells were transduced with lentivirus-mediated Scr control, shC1QBP or pCDH control, pCDH-C1QBP, and the expression of C1QBP was examined by western blot (A) and qRT-PCR (B). (C) According to the manufacturer's instructions 10⁷ cells in each group were collected and froze with liquid nitrogen. Metabonomic techniques were used to detect the differences of 200 major metabolites involved in cell metabolism in RCC 786-O cells control group (pCDH) and C1QBP overexpression group (pCDH-C1QBP), n = 6. (D) Among the 17 differential metabolites, the change of hypoxanthine was the most significant, and decreased by 80% in 786-O cells with overexpression of C1QBP compared with the control group, P = 0.01, n = 6. (E) Hypoxanthine was examined by assay kit in ACHN and 786-O cells with C1QBP knockdown (left panel) and overexpression (right panel), P < 0.001 by t-test, data were presented as mean ± SD from three independent repeats. Naïve: untreated; Scr: down-expression empty plasmid control; shC1QBP: C1QBP knockdown; pCDH: overexpression empty plasmid control; C1QBP/pCDH-C1QBP: C1QBP overexpression. Statistically significant differences were indicated: ***, P < 0.001. NS: no significant difference.

Figure 2

C1QBP regulates the expression of XDH in RCC. (A) Western blot detected the expression of XDH in ACHN and 786-O cells with stable C1QBP knockdown (left panel) and C1QBP overexpression (right panel). (B) The XDH mRNA was examined by qRT-PCR in ACHN (upper panel) and 786-O (lower panel) cells with stable C1QBP knockdown (left panel) and C1QBP overexpression (right panel). The expression of C1QBP and XDH was detected by western blot both in tumors (T) and adjacent normal tissues (N) of 30 primary RCC patients. (C) Representative 8 pairs of western blot images were shown. (D) The expression of C1QBP (left panel) and XDH (right panel) was normalized with β-actin and quantified by Image J software in 30 primary RCC patients. The analysis was used paired t-test, P = 0.015, P < 0.0001, respectively, n = 30. (E) The expression of C1QBP and XDH was examined by immunohistochemistry staining in a study cohort with 57 pairs of RCC (Tumor) and adjacent normal kidney tissues (Para-tumor). Representative pictures were shown, and brown signals indicated positive staining. Scale bar = 50μm. Statistically significant differences were indicated: *, P < 0.05, **, P < 0.01 and ***, P < 0.001.

Figure 3

C1QBP modulates XDH mRNA at translational level. The XDH pre-mRNA was examined by qRT-PCR in control group and C1QBP knockdown or overexpression group of ACHN (A) and 786-O (B) cells. TNF-α, IL6, IL-1β, and IFN-γ mRNA were examined by qRT-PCR in control group and C1QBP knockdown or overexpression group of ACHN (C) and 786-O (D) cells. (E) Control and C1QBP overexpressed ACHN and 786-O cells were treated with 5 μg/ml actinomycin D at indicated time point 0 min, 15 min, 30 min, 45 min, and 60 min. The RNA was extracted and XDH mRNA was detected by qRT-PCR. Statistically significant differences were indicated: *, P < 0.05, **, P < 0.01 and ***, P < 0.001. NS: no significant difference.

Figure 4

C1QBP is critical for ROS level and apoptosis of RCC cells. (A) In ACHN and 786-O RCC cells, ROS was examined and quantified by detecting fluorescence intensity of DCF and normalizing to control. Left panel: control and C1QBP knockdown group, right panel: control and C1QBP overexpression group. In ACHN and 786-O RCC cells, the percentage of annexin V-positive cells was determined by flow cytometry (B and D). Quantitative analysis of the apoptotic rate of cells with C1QBP knockdown (C) and C1QBP overexpression (E) was performed by normalizing to the corresponding control group. (F) The expression of C1QBP, XDH, cleaved-caspase-3, bcl2, and bax was examined by western blot in ACHN and 786-O cells with controls and C1QBP knockdown and overexpression. Data were presented as mean ± SD by t-test, n = 3; *, P < 0.05, **, P < 0.01, and ***, P < 0.001 compared to the control (Scr or pCDH).

Figure 5

XDH is critical for C1QBP-regulated ROS production and apoptosis of RCC. XDH knockdown by using three independent XDH siRNAs (si-XDH-1, si-XDH-2, si-XDH-3) in ACHN and 786-O cells were evidenced by qRT-PCR (A) and western blot (B). ACHN and786-O cells were transfected with pCDH + si-NC, pCDH-C1QBP + si-NC, pCDH-C1QBP + si-XDH, and then ROS level (C and D) and apoptosis (E and F) were examined by flow cytometry, and (G) the expression of C1QBP, XDH, cleaved-caspase-3, bcl2, and bax was examined by western blot. β-actin was used as an internal control. Data were presented as mean ± SD by t-test. Statistically significant differences were indicated: *, P < 0.05, **, P < 0.01 and ***, P < 0.001. NS: no significant difference.

Figure 5

XDH is critical for C1QBP-regulated ROS production and apoptosis of RCC. XDH knockdown by using three independent XDH siRNAs (si-XDH-1, si-XDH-2, si-XDH-3) in ACHN and 786-O cells were evidenced by qRT-PCR (A) and western blot (B). ACHN and786-O cells were transfected with pCDH + si-NC, pCDH-C1QBP + si-NC, pCDH-C1QBP + si-XDH, and then ROS level (C and D) and apoptosis (E and F) were examined by flow cytometry, and (G) the expression of C1QBP, XDH, cleaved-caspase-3, bcl2, and bax was examined by western blot. β-actin was used as an internal control. Data were presented as mean ± SD by t-test. Statistically significant differences were indicated: *, P < 0.05, **, P < 0.01 and ***, P < 0.001. NS: no significant difference.

Figure 6

C1QBP suppresses RCC tumor growth and regulates the expression of XDH and apoptotic proteins in vivo. ACHN cells transduced with luciferase-labeled C1QBP overexpression (C1QBP-luc) or control (pCDH-luc) were used to implant into BALB/c nude mice for 8 weeks, n = 6 each group. The bioluminescence was detected by the IVIS imaging system and followed by histological examination. (A) Tumor growth was examined by the IVIS imaging system, and representative images were shown. (B) Relative tumor bioluminescence intensity was quantified, n = 4 each group. (C) Left primary renal tumor weight was measured and analyzed through subtracting corresponding weight of right renal, respectively, n = 6. (D) Mice primary renal tumors were collected and processed for IHC staining of C1QBP, XDH, caspase-3, bax, and bcl2 and representative images were shown. Scale bar = 50μm. All data were presented as mean ± SD, *, P < 0.05, ***, P < 0.001 by Student's t-test.