Supplementary Figures 1 through 26, Supplementary Tables 1 through 5, and Supplementary Materials and Methods from Synergistic Simvastatin and Metformin Combination Chemotherapy for Osseous Metastatic Castration-Resistant Prostate Cancer
Authors: Babcook Melissa A. , Shukla Sanjeev , Fu Pingfu , Vazquez Edwin J. , Puchowicz Michelle A. , Molter Joseph P. , Oak Christine Z. , MacLennan Gregory T. , Flask Chris A. , Lindner Daniel J. , Parker Yvonne , Daneshgari Firouz , Gupta Sanjay 
Published: April 2023

<p>Figure S1. HMG-CoAR protein is over-expressed and its degradation dysregulated in omCRPC cell lines. Figure S2. Total cholesterol significantly increased in omCRPC cell lines. Figure S3. Cellular free-to-total cholesterol ratio minimally affected by PCa progression to castration-resistance. Figure S4. (prev. page) AMPK activity is hampered in omCRPC cells through Ser-485/491 phosphorylation by constitutively activated Akt. Figure S5. SIM and MET individual treatment dose-response plots. Figure S6. (prev. page) Synergistic quantification of combination SIM and MET treatment in C4-2B3 and C4-2B4 omCRPC cells using Chou-Talalay method. Figure S7. Viability effect of deuterium oxide on C4-2B3 and C4-2B4 omCRPC cells and standard curves for cholesterol and deuterium quantization. Figure S8. Schematic of in vivo experimental design. Figure S9. Low- and high-dose SIM and MET combination treatment inhibited C4-2B4 primary tumor growth in an in vivo model of CRPC. Figure S10. (prev. page) SIM and MET treatment does not adversely affect mouse body weight. Figure S11. SIM and MET treatment does not increase plasma alanine aminotransferase (ALT) levels. Figure S12. SIM and MET treatment does not induce pathology or injury to liver. Figure S13. SIM treatment does not induce pathology of muscle. Figure S14. SIM lactone-to-beta-hydroxyacid hydrolysis. Figure S15. (prev. page) Statin derivatization. Figure S16. SIM acid total ion chromatograph and mass spectrum. Figure S17. SIM lactone total ion chromatograph and mass spectrum. Figure S18. Rosuvastatin total ion chromatograph and mass spectrum. Figure S19. SIM acid calibration curves. Figure S20. MET and phenformin derivatization. Figure S21. MET total ion chromatograph and mass spectrum. Figure S22. Phenformin total ion chromatograph and mass spectrum. Figure S23. MET calibration curves. Figure S24. Comparable metabolic aberrations within Akt-AMPK-HMG-CoAR network observed in other in vitro models of DTXresistant, hormone-refractory bone metastatic prostate and breast cancer. Figure S25. Synergistic combination SIM and MET significantly more effective than DTX at cell viability inhibition in multiple paradigms of DTX-resistant and hormone refractory osseous metastatic cell lines of prostate and breast cancer. Figure S26. (prev. page) Synergistic quantification of combination SIM and MET in PC-3 D12 DTX-resistant bone metastatic androgen-independent PCa, MDA-MB-231 triple-negative breast cancer, and MDA-MB-231(SA) bone metastatic triple-negative breast cancer cells using Chou-Talalay method. Table S1. Primary antibodies for immunoblotting. Table S2. Concentration of SIM (microM) and MET (mM) alone or in (1:500) combination necessary for cell viability inhibition in C4-2B3 and C4-2B4 omCRPC cells. Fa, fraction of cells affected. Table S3. Concentration of SIM acid and MET in the terminal plasma and ventral prostate (VP) tissue amongst the six mouse treatment groups. Table S4. 1:500 SIM and MET combination synergistically inhibits cell viability in bone metastatic prostate and breast cancer cell lines. Table S5. Concentration of SIM (microM) and MET (mM) alone or in (1:500) combination necessary for cell viability inhibition in PC-3 D12 DTX-resistant bone metastatic androgen-independent PCa cells, MDA-MB-231 triple-negative breast cancer cells, and MDAMB-231(SA) highly bone metastatic triple-negative breast cancer cells. Supplementary Materials and Methods.</p>