Lupeol alters viability of SK-RC-45 (Renal cell carcinoma cell line) by modulating its mitochondrial dynamics

Abstract

Renal cell carcinoma (RCC) is the most common kidney cancer leading to 140,000 deaths per year. Among all RCCs 80% evolve from the epithelial proximal tubular cells within the kidney. There is a high tendency of developing chemoresistance and resistance to radiation therapy in most RCC patients. Therefore, kidney resection is considered as the most effective treatments for patients having localized RCC. There is a high tendency of post-operative recurrence among 20-40% of the patients and this recurrence is not curable. It is also clear that modern medicine has no curative treatment options against metastatic RCC. Lupeol [lup-20(29)-en-3β-ol] is a pentacyclic triterpenoid compound naturally found in various edible fruits and in many traditionally used medicinal plants, and has been demonstrated as effective against highly metastatic melanoma and prostate cancers. The present study was designed to evaluate the effect of lupeol to RCC with molecular details. Treatment with lupeol on SK-RC-45 (a RCC cell line) with the LC₅₀ dose of 40μM (for 48 h) induces mitochondrial hyper fission which eventually leads to apoptosis while SK-RC-45 counteracts by enhancing autophagy-mediated selective removal of fragmented mitochondria. This is the first study which concurrently reports the effects of lupeol on RCC and its effect on the mitochondrial dynamics of a cell. Herein, we conclude that lupeol has potential to be an effective agent against RCC with the modulation of mitochondrial dynamics.

Keywords: Cancer research.

Fig. 1

Structure and effect of Lupeol on the viability of SK-RC-45. Control: vehicle treatment alone (for 48 h); Lupeol (20μM): 20μM Lupeol treatment for 48 h; (a) Structure of Lupeol (b) Shows the dose dependent change in viability (expressed in % over control) after Lupeol exposure for 48 h; (c) Immunoblot analysis of Caspase-3 from whole lysate of SK-RC-45 from all three groups. Note that Lupeol activated Caspase-3 (CSP-3) as evident from increased cleaved expression; (d) FACS analysis of Annexin V-FITC staining. Q1_LL: Annexin V negative cells (normal); Q1_LR: Annexin V positive cells (early apoptosis). Values in % in each quadrant represent the amount of cells in that quadrant. ‘FITC-A’ represents FL1-H filter and ‘Propidium iodide A’ represents FL2-H filter. All data are mean ± SEM. P < 0.05 compared with the control group; Data are represented as the mean ± SEM of three individual experiments. *P < 0.05 vs. Control.

Fig. 2

Effect of Lupeol on the mitochondrial dynamics of SK-RC-45 and its relation with Bcl-2. Control: vehicle treatment alone (for 48 h); Lupeol (20μM): 20μM Lupeol treatment for 48 h; Lupeol (40μM): 40μM Lupeol treatment for 48 h; Control_SK: Skeletonized version of Control from MiNA analysis; Lupeol (20μM): Skeletonized version of Lupeol (20μM) from MiNA analysis; Lupeol (40μM): Skeletonized version of Lupeol (40μM) from MiNA analysis (see the ‘Materials and methods’ for MiNA). (a) Confocal image and respective skeletonized version of representative SK-RC-45. Results of MiNA analysis (b) Networks (this is the number of objects in the image that contain at least 1 junction pixel and are thus comprised of more than one branch), (c) Mean Network Length (The average length of all rods/branches), (d) Mean Network Size (This is the mean number of branches per Network) and (e) Mitochondrial Footprint (This is the total area in the image consumed by signal after being separated from the background that is proportionate with the actual mitochondrial mass). (f) Effect of Bcl-2 knock down on Lupeol's efficacy. (g) RT PCR of Bcl-2 to confirm the knockdown of Bcl-2 m RNA after treatment of cells with Bcl-2 si RNA. Control (si): Control scramble si-RNA treated cells; Control (si)+Lupeol: Control scramble si-RNA treated cells with 40μM Lupeol treatment for 48 h; si-Bcl-2: Bcl-2 si-RNA treated cells; si-Bcl-2+Lupeol: Bcl-2 si-RNA treated cells with 40μM Lupeol treatment for 48 h control. Data are represented as the mean ± SEM of three individual experiments. *P < 0.05 vs. Control. #P < 0.05 vs. Lupeol n. s. indicates ‘non-significant’ difference.

Fig. 3

SK-RC-45's response in respect to Lupeol treatment. Control: vehicle treatment alone (for 48 h); Lupeol (20μM): 20μM Lupeol treatment for 48 h; Lupeol (40μM): 40μM Lupeol treatment for 48 h. (a) Immunoblot analysis of Beclin-1and LC-3B I/II from whole lysate of SK-RC-45 from all three groups. Note that, upon Lupeol treatment cellular Beclin-1 and LC-3B II level (thus promotes LC-3B I to LC-3B II conversion) increased. (b) Effect of 3-MA pretreatment on Lupeol treatment. Note that, upon 3-MA pre-treatment cell death significantly induced upon Lupeol treatment in respect to Lupeol treatment alone. Here Lupeol was used at a dose of 40 μM for 48 h *P < 0.05 vs. Control.