Chaperonin-containing tailless complex polypeptide 1 subunit 6A negatively regulates autophagy and protects colorectal cancer cells from cisplatin-induced cytotoxicity

Abstract

Background: As a member of the chaperonin-containing tailless complex polypeptide 1 (TCP1) complex, which plays a pivotal role in ensuring the accurate folding of numerous proteins, chaperonin-containing TCP1 subunit 6A (CCT6A) participates in various physiological and pathological processes. However, its effects on cell death and cancer therapy and the underlying mechanisms need further exploration in colorectal cancer (CRC) cells.

Aim: To explore the effects of CCT6A on cell death and cancer therapy and the underlying mechanisms in CRC.

Methods: Cell proliferation was evaluated using the MTS assay, EdU staining, and colony growth assays. The expression of CCT6A was monitored by immunoblotting and quantitative PCR. CCT6A was knocked out by CRISPR-Cas9, and overexpressed by transfecting plasmids. Autophagy was examined by immunoblotting and the mCherry-GFP-LC3 assay. To monitor apoptosis and necroptosis, immunoblotting, co-immunoprecipitation, and flow cytometry were employed.

Results: Cisplatin (DDP) exerted cytotoxic effects on CRC cells while simultaneously downregulating the expression of CCT6A. Depletion of CCT6A amplified the cytotoxic effects of DDP, whereas overexpression of CCT6A attenuated these adverse effects. CCT6A suppressed autophagy, apoptosis, and necroptosis under both basal and DDP-treated conditions. Autophagy inhibitors significantly enhanced the cytotoxic effects of DDP, whereas a necroptosis inhibitor partially reversed the cell viability loss induced by DDP. Furthermore, inhibiting autophagy enhanced both apoptosis and necroptosis induced by DDP.

Conclusion: CCT6A negatively modulates autophagy, apoptosis, and necroptosis, and CCT6A confers resistance to DDP therapy in CRC, suggesting its potential as a therapeutic target.

Keywords: Autophagy; Chaperonin-containing tailless complex polypeptide 1 subunit 6a; Cisplatin; Colorectal cancer; Necroptosis.

Figure 1

Cisplatin reduces cell proliferation in colorectal cancer. A and B: Indicated concentrations of cisplatin (DDP) (μg/mL) were used to treat HT29 and SW480 cells, and the cell viability was measured by MTS assay; C and D: For the colony growth assay, HT29 and SW480 were treated with the indicated concentration gradient of DDP (μg/mL), and colony formation numbers were calculated and graphically represented using ImageJ and Prism GraphPad software. Scale bar = 1 cm; E and F: EdU staining assay was conducted to evaluate cell proliferation following DDP treatment (5 μg/mL, hereafter unless otherwise indicated) for 3 hours. Scale bar = 0.5 mm. ^bP < 0.01 vs control (Ctrl).

Figure 2

Cisplatin decreases the protein level of chaperonin-containing tailless complex polypeptide 1 subunit 6a. A: Quantitative PCR was conducted to detect the transcription level of chaperonin-containing tailless complex polypeptide 1 subunit 6a (CCT6A) in both HT29 and SW480 cells following cisplatin (DDP) treatment for 4 hours; B and C: Following treatment with DDP for the indicated period, the whole protein was extracted and subjected to immunoblot analysis with the indicated antibodies. ^bP < 0.01 vs control (Ctrl).

Figure 3

Chaperonin-containing tailless complex polypeptide 1 subunit 6a rescues the cell viability loss induced by cisplatin. A and B: The knockout (KO)-chaperonin-containing tailless complex polypeptide 1 subunit 6A (CCT6A) HT29 cells and overexpression (OE)-CCT6A SW480 cells were constructed as described in Methods, and the efficacies were confirmed by immunoblotting and immunofluorescence, respectively. Scale bar = 20 μm; C and D: Utilizing the MTS assay, the cell viability was identified in the indicated cells in the presence or absence of cisplatin (DDP); E-H: Cell proliferation in the indicated cells with or without DDP (μg/mL) treatment was analyzed by colony growth assay (scale bar = 1 cm); I-K: EdU staining assay was performed in the indicated cells with or without DDP treatment (scale bar = 0.5 mm). ^aP < 0.05 vs control (Ctrl); ^bP < 0.01 vs Ctrl.

Figure 4

Chaperonin-containing tailless complex polypeptide 1 subunit 6A affects the autophagy pathway in colorectal cancer cells. A and B: Cell lysates were extracted from knockout (KO)-Control (Ctrl) and KO-chaperonin-containing tailless complex polypeptide 1 subunit 6A (CCT6A) HT29 cells, as well as overexpression (OE)-Ctrl and OE-CCT6A SW480 cells, and then subjected to immunoblotting using the indicated antibodies; C: Following treatment with chloroquine diphosphate salt (CQ, 20 μM, hereafter unless otherwise indicated) for 2 hours, immunoblotting was performed with the total proteins extracted from the indicated cells. DDP: Cisplatin; LC3: Light chain 3; mTOR: Mammalian target of rapamycin; Ulk1: Unc-51-like autophagy activating kinase 1.

Figure 5

Chaperonin-containing tailless complex polypeptide 1 subunit 6A negative modulates autophagy induced by cisplatin in colorectal cancer. A-C: Cells were treated with or without cisplatin (DDP) in the presence or absence of CQ for 2 hours, cell lysates were extracted and performed immunoblotting with indicated antibodies; D-G: Following transfection with mCherry-GFP-LC3B adenovirus for 24 hours, cells were split onto coverslips, and then captured by immunofluorescence microscopy (1000 magnification; green arrow: green fluorescence protein (GFP) dot; red arrow: MCherry dot; yellow arrow: Merge dot; white arrowhead: Sole red dot; scale bar = 20 μm). ^aP < 0.05 vs control (Ctrl); CCT6A: Chaperonin-containing tailless complex polypeptide 1 subunit 6a; CQ: Chloroquine diphosphate salt; KO: Knockout; OE: Overexpression.

Figure 6

Chaperonin-containing tailless complex polypeptide 1 subunit 6A negatively modulates apoptosis and necroptosis in colorectal cancer. A: After treating the indicated cells with cisplatin (DDP) for 24 hours, cell lysates were prepared and subjected to immunoblotting; B and C: Following treatment of the cells with DDP for 24 hours, the apoptosis were determined by flow cytometry (Annexin V-positive), and the data were visualized in histograms; D: Following treatment with DDP for 24 hours, cell lysates were performed immunoblotting with indicated antibodies; E and F: Cells were exposed to the appropriate drugs for 24 hours (necrostatin-1 [Nec-1]: 10 μM, hereafter unless otherwise indicated), cells were lysed, and immunoprecipitated using the receptor-interacting protein kinase 3 (RIP3) antibody. The immunoprecipitates were resolved by electrophoresis and probed by immunoblotting with the indicated antibodies; G: Cell viability in knockout (KO)-Control (Ctrl) and KO-chaperonin-containing tailless complex polypeptide 1 subunit 6a (CCT6A) cells was analyzed following the indicated treatments for 24 hours. ^bP < 0.01 vs control. Bcl-xL: B-cell lymphoma-extra large; IgG: Immunoglobulin G (negative control antibody); MLKL: Mixed lineage kinase domain-like; Nec-1; Necrostatin-1; OE: Overexpression; PARP-1; Poly(ADP-ribose) polymerase 1; TH: Total homogenate.

Figure 7

Inhibition of autophagy enhances the cytotoxicity of cisplatin. A-D: The MTS assay was conducted to assess the cell viability after applying the specified treatments for 24 hours (3-Methyladenine [3-MA]: 2 mM) (A and C), colony growth assay was performed with indicated treatments (cisplatin [DDP]: 1 μM; chloroquine diphosphate salt [CQ]: 5 μM; 3-MA: 0.5 mmol/L) (scale bar = 1 cm) (B and D); E: Following appropriate treatments for 24 hours, cell lysates were performed immunoblotting with indicated antibodies; F: Co-immunoblotting assay was conducted with the receptor-interacting protein kinase 3 antibody, and then subjected to immunoblotting; G: Schematic model of chaperonin-containing tailless complex polypeptide 1 subunit 6a (CCT6A) modulates the cytotoxicity of DDP via regulating autophagy. ^bP < 0.01 vs control (Ctrl). Bcl-xL: B-cell lymphoma-extra large; IgG: Immunoglobulin G (negative control antibody); MLKL: Mixed lineage kinase domain-like; Nec-1; Necrostatin-1; PARP-1; Poly(ADP-ribose) polymerase 1; RIP1: Receptor-interacting protein kinase 1; TH: Total homogenate.