Shikonin Inhibits Cell Growth of Sunitinib-Resistant Renal Cell Carcinoma by Activating the Necrosome Complex and Inhibiting the AKT/mTOR Signaling Pathway

Figures

• 

  Figure 1. Growth of parental and sunitinib-resistant Caki-1 (a,b), 786-O (d,e), KTCTL-26 (g,h), and A-498 (j,k) cells after 24, 48, and 72 h exposure to SHI (0.5–2.5 µM). Cell number was set to 100% after 24 h incubation. Error bars indicate standard deviation (SD). Significant difference to untreated control: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n = 5. Proliferation of parental and sunitinib-resistant Caki-1 (c), 786-O (f), KTCTL-26 (i), and A-498 (l) after 48 h incubation with SHI (0.5–2.5 µM). Untreated controls were set to 100%. Error bars indicate standard deviation (SD). Significant difference to untreated controls: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n = 4.

• 

  Figure 2. Clonogenic growth of parental and sunitinib-resistant Caki-1 (a), 786-O (b), KTCTL-26 (c), and A-498 (d) cells treated with SHI (0.1–0.5 µM) for 10 days. Untreated cells served as controls (set to 100%). Representative images of the clonogenic assays are shown on the right. Error bars indicate standard deviation (SD). Significant difference to untreated controls: * p ≤ 0.01, ** p ≤ 0.01, *** p ≤ 0.001. n = 5.

• 

  Figure 3. Cell cycle proportionality of parental and sunitinib-resistant Caki-1 (a), 786-O (b), KTCTL-26 (c), and A-498 (d) cells in G0/G1, S, and G2/M phases following 48 h exposure to 1.5 µM SHI. Untreated controls set to 100% (dotted line). Error bars indicate standard deviation (SD). Significant difference to untreated control: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n = 5.

• 

  Figure 4. Protein expression and activity of cell cycle regulating proteins in parental and sunitinib-resistant Caki-1 cells after 48 h exposure to 1 or 1.5 µM SHI (a). Representative Western blot analysis (n = 4). The housekeeping protein, β-actin, served as internal control. Pixel density analysis (Western blot) of the cell cycle regulating proteins p21 (b), p27 (c), cyclin A (d), cyclin B (e), CDK1 (f), pCDK1 (g), and CDK2 (h) in parental and sunitinib-resistant Caki-1 cells after 48 h exposure to 1.5 µM SHI, compared to untreated control (set to 100%). Protein analysis was performed with respect to the housekeeping protein β-actin. n.d. = not detectable. Error bars indicate standard deviation (SD). Significant difference to untreated control: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n = 4. For detailed information regarding the Western blots, see Figure S1.

• 

  Figure 5. Protein expression and activity of cell cycle regulating proteins in parental and sunitinib-resistant 786-O cells after 48 h exposure to 1 or 1.5 µM SHI (a). Representative Western blot analysis (n = 4). The housekeeping protein β-actin served as internal control. Pixel density analysis (Western blot) of the cell cycle regulating proteins p21 (b), p27 (c), cyclin A (d), cyclin B (e), CDK1 (f), pCDK1 (g), CDK2 (h), and pCDK2 (i) in parental and sunitinib-resistant 786-O cells after 48 h exposure to 1.5 µM SHI, compared to untreated control (set to 100%). Each protein analysis was accompanied and normalized by the housekeeping protein β-actin. Error bars indicate standard deviation (SD). Significant difference to untreated control: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n = 4. For detailed information regarding the Western blots, see Figure S2.

• 

  Figure 6. Apoptosis in parental and sunitinib-resistant Caki-1 (a), 786-O (b), KTCTL-26 (c), and A-498 (d) cells treated with 1.5 µM SHI for 48 h. Untreated cells served as controls (set to 100%). Error bars indicate standard deviation (SD). Significant difference to untreated control: * p ≤ 0.01, ** p ≤ 0.01, *** p ≤ 0.001. n = 3.

• 

  Figure 7. Necroptosis in parental and sunitinib-resistant Caki-1 (a,b), 786-O (c,d), KTCTL-26 (e,f), and A-498 (g,h) cells treated with 0.5–2.5 µM SHI and 80 µM necrostatin-1 (Nec-1), a necroptosis inhibitor, for 24 h. Error bars indicate standard deviation (SD). Bracketed asterisks indicate a significant difference between Nec-1 untreated and treated cells, unbracketed asterisks indicate significant difference to controls, untreated with SHI (set to 100%): * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n = 5.

• 

  Figure 8. Caspase-dependent or -independent cell death induction in parental and sunitinib-resistant Caki-1 (a,b) and 786-O (c,d) cells treated for 24 h with SHI (0.5–2.5 µM) alone or in combination with the multi-caspase inhibitor zVAD (20 µM). Untreated cells served as controls (set to 100%). Error bars indicate standard deviation (SD). Significant difference compared to untreated controls, except for bracketed asterisks, indicating significant difference between untreated and zVAD-treated cells: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n = 3.

• 

  Figure 9. Caspase-independent cell death induction in parental and sunitinib-resistant Caki-1 (a–c) and 786-O (d–f) cells after SHI (1.0 µM) or zVAD treatment (20 µM) for 6 h. Caspase activity is expressed as percent, with untreated cells serving as controls (set to 100%). Error bars indicate standard deviation (SD). Significance compared to untreated control: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n = 4.

• 

  Figure 10. Necrosome complex key proteins in parental and sunitinib-resistant Caki-1 cells after 6 and 24 h exposure to 1 µM SHI (a). Representative Western blot (n = 3). Pixel density analysis of the necrosome complex proteins RIP1 (b), pRIP1 (c), RIP3 (d), pRIP3 (e), MLKL (f), and pMLKL (g) in parental and sunitinib-resistant cells after 6 and 24 h exposure to 1 µM SHI. Protein analysis was performed by normalization to a total protein control. Untreated cells served as controls (set to 100%). Error bars indicate standard deviation (SD). Significant difference to untreated control: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n = 3. For detailed information regarding the Western blots, see Figure S3.

• 

  Figure 11. AKT/pAKT and mTOR/pmTOR in parental and sunitinib-resistant Caki-1 (a) and 786-O (b) cells after 48 h exposure to SHI (0.5, 1.0 µM). Representative Western blot (n = 4). Pixel density analysis (Western blot) of AKT (c,g), pAKT (d,h), mTOR (e,i), and pmTOR (f,j), compared to untreated controls (set to 100%). Pixel density was normalized by a total protein staining. Error bars indicate standard deviation (SD). Significant difference to untreated control: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. n = 4. For detailed information regarding the Western blots, see Figure S4.

• 

  Figure 12. Functional relevance of AKT: growth of parental and sunitinib-resistant Caki-1 (a,b) and 786-O (c,d) cells after 48 h incubation with 0.5–2.0 µM SHI or 20 µM AZD5363 (AKT inhibitor). Untreated cells served as control (= 100%). Error bars indicate standard deviation (SD). Significant difference to untreated control: * p ≤ 0.05, *** p ≤ 0.001. n = 3.