Somatostatin derivative (smsDX) targets cellular metabolism in prostate cancer cells after androgen deprivation therapy

Abstract

Cancer cell metabolism responsive to androgen deprivation therapy (ADT) may be involved in the development and progression of prostate cancer and the ultimate failure of androgen-deprivation therapy. To investigate the metabolism regulation effects on androgen-independent growth of prostate cancer, an established LNCaP-s cell model that resembles the clinical scenario of castration-resistant prostate cancer (CRPC), was used in this current study. This cell line was cultured from androgen-sensitive LNCaP parental cells, in an androgen-reduced condition, resembling clinical androgen deprivation therapy. To assess the effects of smsDX on the invasiveness of prostate cancer cells we used wound healing assay and Matrigel™ invasion assay. We evaluated differentially expressed proteins of the parental LNCaP cells and LNCaP-s cells after ADT by means of two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF mass spectrometric analysis. The covered area in the wound and the number of cells invading through a Matrigel chamber were significantly smaller for cells treated with smsDX than they were for control cells treated with vehicle. 56 proteins were found differentially expressed in LNCaP-s cells compared to LNCaP cells, majority of them were down-regulated after ADT treatment. 104 proteins of LNCaP cells and 86 in LNCaP-s cells, separately, were found differentially expressed after treatment with smsDX, When we explored these protein functions within the website UniProtKB/Swiss-Prot, surprisingly, most of the proteins were found to be involved in the cellular metabolism and mitochondrial function regulation. LNCaP-s as potential metastatic androgen-independent cancer cells, its metabolism and mitochondrial functions could be altered by a new somatostatin derivative smsDX, the smsDX regulatory effects on metabolism in LNCaP-s deliver more therapeutic information with the treatment of CRPC.

Figure 1. Characterizition of LNCaP-s cells.

Parental LNCaP cells have an epitheial morphology and LNCaP-s cells showed a neuronal morphology. AR, CgA and NSE expressions in LNCaP and LNCaP-s cells examined by RT-PCR and Western Blotting.

Figure 2. Inhibition of cell migration of prostate cancer cells LNCaP/LNCaP-s by smsDX.

Representative photomicrographs demonstrate wound closure in LNCaP cells (A) and LNCaP-s cells (B). Monolayers of LNCaP/LNCaP-s cells were disrupted with sterile pipette tip to create uniformand treated with PBS or 1–10 nM smsDX for 24 hours.

Figure 3. Inhibition of cell invasiveness of prostate cancer cells LNCaP/LNCaP-s by smsDX.

Matrigel invasion data when LNCap/LNCaP-s cells in upper well were incubated in serum-free medium and lower well was filled with serum-free medium and 1–10 nM smsDX or PBS. After 24 hours, number of cells that invaded through Matrigel was counted in at least 10 fields per well. Representative photographs reveal LNCaP (A) and LNCaP-s (B) cells that invaded through Matrigel. Reduced from ×100.

Figure 4. Representative examples of 2-DE gels.

Whole-cell lysate was subjected to 2-DE using IPG strips pH 3–10 in the first and 12.5% SDS polyacrylamide gel in the second dimension. A, Parental LNCaP cells, B, derivative LNCaP-s cells. C, LNCaP+smsDX , D, LNCaP-s+smsDX.

Figure 5. Validation of selected proteins by using western blotting.

A, Lower expressions of protein TCTP, STMN1, and CXB3 in LNCaP cells after ADT in LNCaP-s cells. B, Down regulated expressions of TOM40, GRP78 and VDAC2 in LNCaP cells by androgen-deprivation, up regulated expressions in LNCaP-s cells by smsDX treatment.

Figure 6. Metabolic pathways regulated by somatostatin dervivative (smsDX) in prostate cancer cells.

Enzymes and metabolites that are part of glycosis, fatty acide synthesis and the TCA cycle are shown. G6PD, Glucose-6-phosphate 1-dehydrogenase. ENO1, Alpha enolase. PPP, pentose phosphate pathway. ACAT2, Acetyl-CoA acetyltransferase. ACAMD, Acyl-CoA dehydrogenase. IDH2, Isocitrate dehydrogenase [NADP]. MDHM, Malate dehydrogenase. GLUD1, Glutamate dehydrogenase1. α-KG, α-Ketoglutarate. This simplified diagram is based on the KEGG pathway database (http://www.genome.jp/kegg/pathway.html). The enzymes in circle were identified by 2-DE MS in this current study.