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. 2016 Jun 29:4:e2168.
doi: 10.7717/peerj.2168. eCollection 2016.

The chemomodulatory effects of glufosfamide on docetaxel cytotoxicity in prostate cancer cells

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The chemomodulatory effects of glufosfamide on docetaxel cytotoxicity in prostate cancer cells

Reem T Attia et al. PeerJ. .

Abstract

Background. Glufosfamide (GLU) is a glucose conjugate of ifosfamide in which isophosphoramide mustard is glycosidically linked to the β-D-glucose molecule. Based on GLU structure, it is considered a targeted chemotherapy with fewer side effects. The main objective of the current study is to assess the cytotoxic potential of GLU for the first time in prostate cancer (PC) cells representing different stages of the tumor. Furthermore, this study examined the potential synergistic activity of GLU in combination with docetaxel (DOC). Methods. Two different cell lines were used, LNCaP and PC-3. Concentration-response curves were assessed. The tested groups per cell line were, control, GLU, DOC and combination. Treatment duration was 72 h. Cytotoxicity was assessed using sulforhodamine B (SRB) assay and half maximal inhibitory concentration (IC50) was calculated. Synergy analyses were performed using Calcusyn(®)software. Subsequent mechanistic studies included β-glucosidase activity assay, glucose uptake and apoptosis studies, namely annexin V-FITC assay and the protein expression of mitochondrial pathway signals including Bcl-2, Bax, Caspase 9 and 3 were assessed. Data are presented as mean ± SD; comparisons were carried out using one way analysis of variance (ANOVA) followed by Tukey-Kramer's test for post hoc analysis. Results. GLU induced cytotoxicity in both cell lines in a concentration-dependent manner. The IC50 in PC-3 cells was significantly lower by 19% when compared to that of LNCaP cells. The IC50 of combining both drugs showed comparable effect to DOC in PC-3 but was tremendously lowered by 49% compared to the same group in LNCaP cell line. β-glucosidase activity was higher in LNCaP by about 67% compared to that determined in PC-3 cells while the glucose uptake in PC-3 cells was almost 2 folds that found in LNCaP cells. These results were directly correlated to the efficacy of GLU in each cell line. Treatment of PC cells with GLU as single agent or in combination with DOC induced significantly higher apoptosis as evidenced by Annexin V-staining. Apoptosis was significantly increased in combination group by 4.9 folds and by 2.1 Folds when compared to control in LNCaP cells and PC-3 cells; respectively. Similarly, the expression of Bcl-2 was significantly decreased while Bax, caspase 9 and 3 were significantly increased in the combined treatment groups compared to the control. Conclusion. GLU has a synergistic effect in combination with DOC as it increases the cell kill which can be attributed at least partially to apoptosis in both the tested cell lines and it is suggested as a new combination regimen to be considered in the treatment of the prostate cancer. Further experiments and clinical investigations are needed for assessment of that regimen.

Keywords: Apoptosis; Docetaxel; Glufosfamide; LNCaP; PC-3; Prostate cancer.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Concentration response curves.
(A) The effect of Glufosfamide, Docetaxel, combination on PC-3 cells. (B) The effect of Glufosfamide, Docetaxel, combination on LNCaP cells. Data are means ± SD (n = 6). Experiments were done in triplicates.
Figure 2
Figure 2. Synergy analysis curve for GLU/DOC combinations in (A) LNCaP PC cells, (B) PC-3 cells.
Graph was plotted using calcusyn software.
Figure 3
Figure 3. Levels of glucose uptake in U87, PC-3 and LNCaP cell lines.
The levels were measured by glucose uptake assay kit using fluorescent glucose 2-NBDG. Data are represented as mean ± SD. (∗∗∗) significantly different from U87 at P < 0.001. (##) significantly different from PC-3 at P < 0.01. Tests were done in triplicates.
Figure 4
Figure 4. Beta-glucosidase activity in U87, PC-3 and LNCaP cells measured by beta-glucosidase assay kit using the 2-NPG substrate.
Data are represented as mean ± SD. (∗∗∗) significantly different from U87 at P < 0.001. (##) signifcantly different from PC-3 at P < 0.01.
Figure 5
Figure 5. (A) AnnexinV-FITC apoptosis assay for LNCaP cells after treatment for 72 h.
The experiment was done in triplicates and a representative figure was chosen for the dot plot. (B) AnnexinV-FITC apoptosis assay for PC-3 cells after treatment for 72 h the experiment was done in triplicates and a representative figure was chosen for the dot plot. (C) Effect of different treatments on the Annexin V-FITC Positive staining in PC-3 and LNCaP Cells. The experiment was done in triplicates. (∗∗∗) significantly different compared to the control at P < 0.001. (∗∗) significantly different compared to the control at P < 0.01.). () significantly different compared to GLU at P < 0.001. (ΔΔ) significantly different compared to GLU at P < 0.01. (###) Significantly different than DOC at P < 0.001. (#) Significantly different than DOC at P < 0.05. Data are means ± SD (n = 3). Data are means ± SD (n = 3).
Figure 6
Figure 6. (A) The effect of GLU, DOC and their combination on the expression of mitochondrial apoptosis signaling proteins in PC-3 prostate cancer cells.
() Significantly different than the control at P < 0.05 (∗∗) significantly different compared to the control at P < 0.01. (∗∗∗) significantly different compared to the control at P < 0.001. (Δ) significantly different compared to GLU at P < 0.05. (ΔΔ) significantly different compared to GLU at P < 0.01. (ΔΔΔ) significantly different compared to GLU at P < 0.001. (#) Significantly different than DOC at P < 0.05. (##) significantly different compared to DOC at P < 0.01. (###) significantly different compared to DOC at P < 0.00. Data are means ± SD (n = 3) of relative band densities normalized to β-actin content. (B) The effect of GLU, DOC and their combination on the expression of many cell survival and apoptotic pathways in LNCaP prostate cancer cells. () Significantly different than the control at P < 0.05 (∗∗) significantly different from the control at P < 0.01. (∗∗∗) significantly different compared to the control at P < 0.001. (Δ) significantly different compared to GLU at P < 0.05. (ΔΔ) significantly different compared to GLU at P < 0.01. (ΔΔΔ) significantly different compared to GLU at P < 0.001. (#) Significantly different than DOC at P < 0.05. (##) significantly different compared to DOC at P < 0.01. (###) significantly different compared to DOC at P < 0.001. Data are means ± SD (n = 3) of relative band densities normalized to β-actin content.

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References

    1. Arafa HM. Possible contribution of beta-glucosidase and caspases in the cytotoxicity of glufosfamide in colon cancercells. European Journal of Pharmacology. 2009;616(1–3):58–63. doi: 10.1016/j.ejphar.2009.06.024. - DOI - PubMed
    1. Baker J, Ajani J, Scotte F, Winther D, Martin M, Aapro MS, Von Minckwitz G. Docetaxel-related side effects and their management. European Journal of Oncology Nursing. 2009;13(1):49–59. doi: 10.1016/j.ejon.2008.10.003. - DOI - PubMed
    1. Becker R, Ritter A, Eichhorn U, Lips J, Bertram B, Wiessler M, Zdzienicka MZ, Kaina B. Induction of DNA breaks and apoptosis in crosslink-hypersensitive v79 cells by the cytostatic drug beta-D-glucosyl-ifosfamide mustard. British Journal of Cancer. 2002;86(1):130–135. doi: 10.1038/sj.bjc.6600027. - DOI - PMC - PubMed
    1. Beckner ME, Fellows-Mayle W, Zhang Z, Agostino NR, Kant JA, Day BW, Pollack IF. Identification of ATP citrate lyase as a positive regulator of glycolytic function in glioblastomas. International Journal of Cancer. 2009;126(10):2282–2295. - PMC - PubMed
    1. Beckner ME, Gobbel GT, Abounader R, Burovic F, Agostino NR, Laterra J, Pollack IF. Glycolytic glioma cells with active glycogen synthase are sensitive to PTEN and inhibitors of PI3K and gluconeogenesis. Laboratory Investigation. 2005;85(12):1457–1470. - PubMed

Grants and funding

Dr. Uday Kompella partially supported this research at Colorado University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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