الفهرس 
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Abstract
Prostate cancer (PC) is among the most frequently diagnosed solid tumors in men aside from skin cancer and it is the second leading cause of cancer death in men after lung cancer. Castaration-resistant prostate cancer (CRPC) is an advanced stage of the disease with poor prognosis. The curative treatment for this late stage of PC is still being investigated, yet, the options are limited and this stage of PC is characterized by high morbidity and mortality rates. Although, DOC based chemotherapy was proved to be the first line treatment for that late stage, but still high doses have severe toxic reactions. That is the reason for the urgent need for safer treatment modalities for CRPC patients. DOC remains one of the most effective agents for prolonging survival and improving quality of life of metastatic CRPC. GLU is the glucose conjugate of ifosfamide, in which isophosphoramide mustard (IPM), the bioactive alkylating metabolite of ifosfamide, is covalently linked to β-D-glucose. On entry into cells, the active moiety IPM is thought to be released by either spontaneous hydrolysis or hydrolysis catalyzed by intracellular glucosidases. This makes GLU- targeted therapy in favor of cancer cells over normal ones.
This study was conducted in order to assess the cytotoxic potential of GLU in PC cells namely; androgen-dependent LNCaP and androgen-refractory PC-3 cells. Beta-glucosidase activity and glucose uptake were addressed to unravel the reasons behind the possible comparative toxicity of GLU in both cell lines. DOC cytotoxicity was also elucidated. Combination regimen of both drugs was tested in both cell lines.
Many facets of apoptotic markers were investigated as possible mechanism for the cytotoxicity. The test battery comprised the flowcytometric analysis of the surface marker; Annexin V, Western blotting of caspases 3, 8, 9 and Bcl-2 beside its associated X protein; Bax. Summarized as follows:
GLU induced cytotoxicity in both cell lines; PC-3 and LNCaP in a concentration-dependent manner. It showed more cytotoxicity in the aggressive stage IV PC cell line (PC-3) than the androgen-dependent (LNCaP).
DOC alone showed dose-dependent cell kill but, with more cytotoxicity in LNCaP cell line than PC-3 cell line.
Beta-glucosidase activity in the cell lysates of both cell lines showed higher enzyme activity in LNCaP cell line than PC-3 cell line.
Fluorometric analysis of glucose uptake in both cell lines showed higher glucose uptake in PC-3 cells when compared to the LNCaP cells.
Combined administration of GLU and DOC exhibited synergistic cytotoxicity in both cell with LNCaP cells being more sensitive than PC-3.
Double staining of both cells lines with Annexin V and PI upon treatment of the cells for 72 h was done.
In LNCaP cells, GLU increased the frequency of apoptosis compared to the control.
DOC showed significant increase in apoptosis compared to both untreated control and GLU- treated cells.
GLU augmented DOC’s apoptosis significantly in LNCaP cells compared to control, GLU- and DOC-treated cells.
Western blotting analysis for the pro-apoptotic caspases in LNCaP cells treated with GLU showed a significant increase in caspase 3, 8 and 9 compared to untreated control. In addition, cells treated with DOC had significantly higher expression patterns for caspase 3, 8 and 9 compared to control and GLU-treated cells.
Likewise, combining DOC and GLU provoked a significant increase in caspase 3, 8 and 9 expression compared to control, and GLU- and DOC-treated cells.
Western blotting analysis of the anti-apoptotic Bcl-2 in LNCaP cells showed lower expression in GLU-treated cells compared to untreated control.
DOC- challenged cells showed decrease in Bcl-2 expression, which is significant compared to control and GLU-treated cells.
Similarly, combining the two agents showed significant decrease in Bcl-2 expression compared to control, GLU and DOC treated cells
Bax expression in LNCaP cells was higher following GLU challenge compared to untreated control.
DOC-treated cells showed significant increase in Bax expression compared to control and GLU-treated cells.
The combination of DOC and GLU showed significant increases in Bax expression compared to control, GLU- and DOC-treated cells.
The Bcl-2/Bax ratio was calculated and the groups were compared to each other showing the same expression trends as the Bcl-2 expression.
Summary and Conclusion
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Based on the broad observations scrutinized from the current study, the following conclusions could be drawn:
1- GLU induced cytotoxicity for the first time in two prostate cancer cell lines namely; androgen-dependent LNCaP and androgen-refractory PC-3 cells in a concentration-dependent manner.
2- The PC-3 cells were more sensitive to GLU than LNCaP cells.
3- β-glucosidase activity in PC-3 cells was less than LNCap cells, while the glucose uptake profile was in favor of the PC-3 cells. Despite the importance of β-glucosidase in the intracellular activation of GLU, glucose uptake of the drug is most probably a rate-limiting step in these cancer cell lines.
4- DOC, as expected, induced cytotoxicity in both cell lines with LNCaP cells being more sensitive to the taxane drug than PC-3 cells.
5- Synergistic cytotoxic interactions were seen in both cell lines; LNCaP cells were more sensitive to this combo.
6- Cytotoxicity of either GLU or DOC in the current study resides, at leat partly, on their apoptotic effects including increasing the surface apoptotic marker; Annexin V, upregulation of apoptotic caspases 3, 8, 9 and downregulation of Bcl-2 with concomitant upregulation of its associated X protein; BAX. Bcl2/Bax ratio was also decreased.
7- Synergistic apoptotic interactions between GLU and DOC were also observed and may account for the synergistic cytotoxic efficacy in both cell lines.
Summary and Conclusion
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8- Irrespective of the mechanisms encountered, the current data may possibly afford an interesting possibility of using GLU to improve the therapeutic benefits of DOC-based chemotherapy in prostate cancers; particularly the refractory types.