Background/Aim: Renal cell carcinoma (RCC) represents about 3% of all diagnosed cancers and in advanced stage is a lethal pathology. Approximately 30% of patients with RCC show metastatic disease at diagnosis and disease recurrence occurs in about 30% of subjects after radical or partial nephrectomy (1). Metastatic RCC (mRCC) is very hard to treat; in fact, subjects with mRCC at 5 years from diagnosis show an overall survival lower than 10% (1). Drug resistance was observed in most patients with mRCC, limiting the efficacy of adjuvant therapy (2). Resistance to targeted drugs is classified into two categories: intrinsic or primary and acquired or secondary resistance (3). Different mechanisms of therapy resistance including the activation of autophagy were identified (2, 3). This biological process may supply energy in case of nutrient deprivation and might trap and destroy therapeutic agents by autophagy vesicles. The aim of the study was to determine whether the inhibition of autophagy can increase the efficacy of conventional chemotherapy. Materials and Methods: Caki-2 and KJ29 clear cell renal cell carcinoma (ccRCC) lines were cultured and treated with desmethylclomipramine (DCMI), an autophagy inhibitor, alone or in combination with the tyrosine kinase inhibitor (TKI) Sunitinib for 48 h. Protein expression was evaluated by western blotting in cells cultured in basal condition and treated with DMCI or Sunitinib alone or in combination. Cell migration was performed after seeding 50,000 KJ29 and Caki-2 cells in 24-well plates and culturing in DMEM/F12 medium supplemented with 10% FBS up to confluence. Next, a groove between the cells was generated using a sterile tip and cells were then grown for 48 h in DMEM/F12 1% FBS in the presence of DCMI (5 μM) and Sunitinib (5 μM) individually or in combination. Cell migration (groove filling) was detected by comparing images acquired at T0 (empty groove) with those acquired after 48 h of culture by a phase contrast microscope equipped with a CCD camera. Apoptosis was evaluated by detecting apoptotic nuclei using Hoechst staining in cells cultured in basal condition or in the presence of DCMI and Sunitinib as described above. After treatment, cells were fixed, permeabilized, and stained with Hoechst 33258 (10 mg/ml) in the dark. Images were acquired at 40× magnification using a Zeiss Axiovert 200 fluorescence microscope equipped with a back-illuminated CCD camera. Statistical analysis was performed using GraphPad Prism software and Anova test. p- Values <0.05 were considered statistically significant. Results: Western blot analysis carried out by using an antibody against the autophagy marker p62 showed that the expression of this protein was significantly increased in both Caki2 and KJ29 cells treated with DMCI compared with untreated cells (Figure 1A). Moreover, the double treatment further enhanced p62 protein content. Notably, when autophagy was activated, p62/SQSTM1 expression was inversely correlated with the levels of autophagy. Interestingly, the inhibition of autophagy by treatment with DMCI reduced the expression of the mesenchymal marker Vimentin in both cell lines (Figure 1A). The combined application of DCMI and Sunitinib seemed to decrease even more Vimentin protein levels. Consistently, the analysis of cell migration by scratch-wound assay showed that the single treatment with DCMI or Sunitinib partially inhibited cell migration, but the combined treatment strongly blocked cell migration in both cell lines (Figure 1B). Finally, the mixed treatment with DCMI and Sunitinib promoted apoptosis in Caki-2 and KJ29 ccRCC cells as compared to untreated cells. In the presence of both compounds, nuclei appeared more condensed, disrupted, and irregular, whereas in control cells, they were rounded (Figure 1C). Conclusion: Drug resistance is a big obstacle for the treatment of advanced tumors including kidney carcinoma. Autophagy activation might be one of the mechanisms responsible for chemotherapy resistance; therefore, this process could be associated with tumor progression and the death of patients. Here, we observed that treatment with the TKI Sunitinib after autophagy inhibition by DMCI reduced more efficiently cell migration compared with Sunitinib alone. Consistently, in ccRCC cells treated with both compounds, a greater reduction of the mesenchymal marked Vimentin was observed. These data indicate that the inhibition of autophagy can increase the efficacy of conventional chemotherapy. Interestingly, the combination of DMCI and Sunitinib also caused the activation of apoptosis; therefore, inhibition of autophagy may increase cell death leading to cancer regression. Taken together, autophagy could be used by cancer cells to remove anticancer drugs, promoting chemotherapy resistance in ccRCC cells. In conclusion, the inhibition of autophagy may represent a new pharmacological strategy for the treatment of metastatic kidney cancer.
THE INHIBITION OF AUTOPHAGY INCREASES SUNITINIB RESPONSE IN KIDNEY CANCER CELLS
Simone PatergnaniMembro del Collaboration Group
;Paolo PintonMembro del Collaboration Group
;Carmelo IppolitoMembro del Collaboration Group
;Gianluca Aguiari
Writing – Original Draft Preparation
2023
Abstract
Background/Aim: Renal cell carcinoma (RCC) represents about 3% of all diagnosed cancers and in advanced stage is a lethal pathology. Approximately 30% of patients with RCC show metastatic disease at diagnosis and disease recurrence occurs in about 30% of subjects after radical or partial nephrectomy (1). Metastatic RCC (mRCC) is very hard to treat; in fact, subjects with mRCC at 5 years from diagnosis show an overall survival lower than 10% (1). Drug resistance was observed in most patients with mRCC, limiting the efficacy of adjuvant therapy (2). Resistance to targeted drugs is classified into two categories: intrinsic or primary and acquired or secondary resistance (3). Different mechanisms of therapy resistance including the activation of autophagy were identified (2, 3). This biological process may supply energy in case of nutrient deprivation and might trap and destroy therapeutic agents by autophagy vesicles. The aim of the study was to determine whether the inhibition of autophagy can increase the efficacy of conventional chemotherapy. Materials and Methods: Caki-2 and KJ29 clear cell renal cell carcinoma (ccRCC) lines were cultured and treated with desmethylclomipramine (DCMI), an autophagy inhibitor, alone or in combination with the tyrosine kinase inhibitor (TKI) Sunitinib for 48 h. Protein expression was evaluated by western blotting in cells cultured in basal condition and treated with DMCI or Sunitinib alone or in combination. Cell migration was performed after seeding 50,000 KJ29 and Caki-2 cells in 24-well plates and culturing in DMEM/F12 medium supplemented with 10% FBS up to confluence. Next, a groove between the cells was generated using a sterile tip and cells were then grown for 48 h in DMEM/F12 1% FBS in the presence of DCMI (5 μM) and Sunitinib (5 μM) individually or in combination. Cell migration (groove filling) was detected by comparing images acquired at T0 (empty groove) with those acquired after 48 h of culture by a phase contrast microscope equipped with a CCD camera. Apoptosis was evaluated by detecting apoptotic nuclei using Hoechst staining in cells cultured in basal condition or in the presence of DCMI and Sunitinib as described above. After treatment, cells were fixed, permeabilized, and stained with Hoechst 33258 (10 mg/ml) in the dark. Images were acquired at 40× magnification using a Zeiss Axiovert 200 fluorescence microscope equipped with a back-illuminated CCD camera. Statistical analysis was performed using GraphPad Prism software and Anova test. p- Values <0.05 were considered statistically significant. Results: Western blot analysis carried out by using an antibody against the autophagy marker p62 showed that the expression of this protein was significantly increased in both Caki2 and KJ29 cells treated with DMCI compared with untreated cells (Figure 1A). Moreover, the double treatment further enhanced p62 protein content. Notably, when autophagy was activated, p62/SQSTM1 expression was inversely correlated with the levels of autophagy. Interestingly, the inhibition of autophagy by treatment with DMCI reduced the expression of the mesenchymal marker Vimentin in both cell lines (Figure 1A). The combined application of DCMI and Sunitinib seemed to decrease even more Vimentin protein levels. Consistently, the analysis of cell migration by scratch-wound assay showed that the single treatment with DCMI or Sunitinib partially inhibited cell migration, but the combined treatment strongly blocked cell migration in both cell lines (Figure 1B). Finally, the mixed treatment with DCMI and Sunitinib promoted apoptosis in Caki-2 and KJ29 ccRCC cells as compared to untreated cells. In the presence of both compounds, nuclei appeared more condensed, disrupted, and irregular, whereas in control cells, they were rounded (Figure 1C). Conclusion: Drug resistance is a big obstacle for the treatment of advanced tumors including kidney carcinoma. Autophagy activation might be one of the mechanisms responsible for chemotherapy resistance; therefore, this process could be associated with tumor progression and the death of patients. Here, we observed that treatment with the TKI Sunitinib after autophagy inhibition by DMCI reduced more efficiently cell migration compared with Sunitinib alone. Consistently, in ccRCC cells treated with both compounds, a greater reduction of the mesenchymal marked Vimentin was observed. These data indicate that the inhibition of autophagy can increase the efficacy of conventional chemotherapy. Interestingly, the combination of DMCI and Sunitinib also caused the activation of apoptosis; therefore, inhibition of autophagy may increase cell death leading to cancer regression. Taken together, autophagy could be used by cancer cells to remove anticancer drugs, promoting chemotherapy resistance in ccRCC cells. In conclusion, the inhibition of autophagy may represent a new pharmacological strategy for the treatment of metastatic kidney cancer.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
