A conjugate of methotrexate and an analog of luteinizing hormone releasing hormone shows increased efficacy against prostate cancer

LHRH receptor, is over-expressed in a variety of human tumors and, is a potential binding site for targeted metastatic prostate cancer therapy. The objectives of our study were to synthesize a bioconjugate of the LHRH analog [DLys6]-LHRH and the anti-tumor agent methotrexate and test the hypothesis that [DLys6]-LHRH-MTX targets and inhibits prostate cancer cell growth in vitro and in vivo. The results of in vitro studies, showed that both [DLys6]-LHRH-MTX and MTX displayed superior cytotoxicity against prostate cancer cells in a concentration-dependent manners, with IC50 concentrations for PC-3 cells of, 1.02 ± 0.18 μmol/L and 6.34 ± 1.01 μmol/L; for DU-145 cells, 1.53 ± 0.27 μmol/L and 8.03 ± 1.29 μmol/L; and for LNCaP cells, 1.93 ± 0.19 μmol/L and 9.68 ± 1.24 μmol/L, respectively. The IC50 values of [DLys6]-LHRH-MTX and MTX were 110.77 ± 15.31 μmol/L and 42.33 ± 7.25 μmol/L, respectively. Finally, [DLys6]-LHRH-MTX significantly improved the anti-tumor activity of MTX in nude mice bearing PC-3 tumor xenografts. The inhibition ratios of tumor volume and tumor weight in the [DLys6]-LHRH-MTX treated group were significantly higher than those in the MTX-treated group. Tumor volume doubling time was also significantly extended from 6.13 days in control animals to 9.67 days in mice treated with [DLys6]-LHRH-MTX. In conclusion, [DLys6]-LHRH -MTX may be useful in treating prostate cancer.

LHRH decapeptides have been used to deliver anti-cancer drugs specifically to cancer cells expressing their receptors. For example, Schally and Nagy 19 used potent LHRH antagonists as targeting moieties for a variety of cancer drugs including doxorubicin (DOX). Studies on the conjugates [DLys 6 ]-LHRH-DOX and [DLys 6 ]-LHRH-2-pyrrolino-DOX showed increased efficacy of doxorubicin, as the LHRH analog maintained its highly targeted binding affinity while the drug retained its cytotoxic effects on the tumor cells 19,20 . Turner and Yates 21 reported that conjugates of lytic peptides and LHRH are very effective in destroying prostate cancer xenografts that express LHRH receptors. Hansel et al. conjugated [DLys 6 ]-LHRH and curcuimn and examined the efficacy of the conjugate ([DLys 6 ]-LHRH-curcumin) against pancreatic cancer cells in vitro and in vivo, their results showed that the conjugate of [DLys 6 ]-LHRH-curcumin was effective at treating pancreatic cancer 22 .
Methotrexate(MTX) was one of the first anti-metabolite drugs used in cancer therapy; this compound, can effectively deactivate the metabolism of diseased cells through programmed cell death and apoptosis 23 . To date, MTX continues to be used extensively in the treatment of various malignancies including childhood acute lymphocytic leukemia, osteosarcoma, non-Hodgkin's lymphoma, Hodgkin's disease, head and neck cancer, lung cancer, prostate cancer and breast cancer 24 . MTX ceases intracellular folate metabolism and finally blocks the synthesis of thymine and purines, leading to theimpairment of tumor growth and induction of cell death by the secondary genotoxic effects or apoptosis. Unfortunately, the development of multidrug resistance in cancer cells significantly restricts the effectiveness of this compound. Thus, the clinical application of MTX has remained limited 25 . To overcome this limitation, in the current study, we conjugated [DLys 6 ]-LHRH and MTX. This article reports the preparation and characterization of [DLys 6 ]-LHRH-MTX using nuclear magnetic resonance (NMR) spectroscopy, and liquid chromatography-mass spectrometry (LC-MS) analysis. Further, it reports the efficacy of the conjugate in vitro in prostate cancer cell lines and in vivo in a mouse PC-3 xenograft model.  6 ]-LHRH-MTX varied for different prostate cancer cell lines: for PC-3, this value was 1.02 ± 0.08 μ mol/L, for DU145, 1.53 ± 0.27 μ mol/L; and for LNCaP 1.93 ± 0.19 μ mol/L; The IC 50 of MTX against the prostate cancer cell lines was as follows: PC-3, 6.34 ± 1.01 μ mol/L, DU145, 8.03 ± 1.29 μ mol/L; and LNCaP 9.68 ± 1.24 μ mol/L; All of the IC 50 values of the different compound are summarized in Table 1. As shown in Fig. 1 26 .

Results and Discussion
To determine the selectivity of LHRH-MTX, we performed a MTT experiment with normal human prostate fibroblast (HPrF) cells. The IC 50 of [DLys 6 ]-LHRH-MTX in HPrF cells was 110.77 ± 15.31 μ mol/L, and the IC 50 of MTX in HPrF cells was 42.33 ± 7.25 μ mol/L. As shown in Table 1  [DLys 6 ]-LHRH-MTX induces apoptosis in prostate cancer cells. To quantitatively investigate apoptotic and early necrotic events in these treated cultures, the fluorescent probe Annexin V-FITC and PI were used, The presence of a apoptotic and early necrotic cells was then determined and quantified by flow cytometry analysis which clearly differentiated normal cells with low Annexin V-FITC and low PI staining, apoptotic cells with high Annexin V-FITC and low PI staining, necrotic cells with high Annexin V-FITC and high PI staining and cell debris with low Annexin VFITC and high PI staining. Figure 4 shows that apoptotic cells were detected after treatment with MTX and [DLys 6 ]-LHRH-MTX for 24 h although the percentage of apoptotic cells varied with each therapy. Cells treated with [DLys 6 ]-LHRH-MTX at 0.98 μ mol/L showed 32.14% early apoptotic (AV+ /PI−) populations and 10.62% late apoptotic/early necrotic (AV+ /PI+ ) cells, while for MTX treatment, these percentages were 15.38% and 8.35%. In addition, it should be noted that the apoptotic and necrotic cell death induced by treatment increased with the drug concentration up to 3.91 μ mol/L, these percentages increased to 47.31% and 14.75% for [DLys 6 ]-LHRH-MTX, and 30.88% and 9.52% for MTX, respectivetly. Figure 4 shows that greater apoptotic cell death was observed following treatment with [DLys 6 ]-LHRH-MTX when compared to MTX alone. Conjugates of [DLys 6 ]-LHRH have previously been shown to exhibit higher rates of apoptosis compared with their unconjugated counterparts 27   likely due to enhanced uptake of MTX via the LHRHR [27][28][29] . Apparently, more early apoptotic cells (Annexin V-FITC+ /PI−) than late apoptotic/early necrotic cells (Annexin V-FITC+ /PI+ ) were observed in cells treated with the two compounds, suggesting progression from early apoptotic to late apoptosis/early necrosis. These results demonstrate that apoptotic cell pathways, but not directly necrotic one, seem to be activated by these compounds.  In addition, as shown in Table 2, the mean weight the of tumor tissues in the [DLys 6 ]-LHRH-MTX treated group was significantly less than that of the saline control group (more than 3-fold less, p < 0.01), the [DLys 6 ]-LHRH group treated group (3-fold less, p < 0.01) and MTX group (approximately 1-fold less, p < 0.05), indicating that LHRH modification further increased the therapeutic efficacy of MTX 22,30,31 . As shown in Fig. 6 the tumor burden was decreased from 81.62 ± 17.12 mg/g body weight in tumor-bearing control animals to 22.65 ± 6.18 mg/g body weight, in animals treated with [DLys 6 ]-LHRH-MTX (P< 0.05 compared with saline control, MTX and [DLys 6 ]-LHRH-saline treated animals). Tumor burden was unchanged in the treatment groups receiving either [DLys 6 ]-LHRH alone or the saline control (Fig. 6). Tumor volume doubling time was also significantly extended from 6.13 days in the control animals to 9.67 days in the mice treated with [DLys 6 ]-LHRH-MTX (Table 2). These results confirm that [DLys 6 ]-LHRH-MTX is indeed more effective than MTX alone in vivo. This efficacy could be attributed to the targeting features of [DLys 6 ]-LHRH-MTX that allow for better localization of treatment doses in tumor tissue and cells compared with the non-targeted treatments.

Evaluation of in vivo
Histologically, the tumor tissue of conjugate-treated animals was found to consist mainly of necrotic cells and fluid. Hematoxylin/eosin stained tumor sections of PC-3 xenografts treated with saline or [DLys 6 ]-LHRH alone showed predominantly viable cells (Fig. 7), whereas tumors from animals treated with [DLys 6 ]-LHRH-MTX showed a high degree of necrosis, when compared to animals treated with the unconjugated lytic peptide and LHRH (Fig. 7). The untreated tumors consisted of sheets of cells with large vesicular hyperchromatic nuclei and prominent nucleoli; many mitotic figures were also seen. In most instances, the tumor margin was rich in blood vessels, which penetrated the rim of the neoplastic tissue. In contrast, the tumors of the LHRH-MTX treated mice were pale and poorly vascularized, although some intact vessels were present within the tissue.

Conclusion
In the study, targeted drug [DLys 6 ]-LHRH-MTX was successfully synthesized and characterized. Upon evaluated in human prostate cancer prostate cancer cell lines (PC-3, DU145 and LNCaP), this targeted drug delivery system displayed a significantly enhanced induction of apoptosis and the inhibition of cell growth activity compared to the non-targeted conjugates. The superior anti-tumor efficacy of [DLys 6 ]-LHRH-MTX was further proveddemonstrated in a PC-3 tumor-bearing mouse model. Our data demonstrate that [DLys 6 ]-LHRH-MTX provides an improved therapeutic index for MTX and may be useful for treatment of LHRH receptor-positive cancers.   (Fig. S1). The mass spectrum of LHRH-MTX is shown in in the supporting information Fig. S1.      cultures were maintained at sub-cofluency at 37 °C in a humidified atmosphere of 5% CO 2 /95% air. All experiments were performed on cells in the logarithmic growth phase.
In the equation, T represents the absorption value of the treatment group; C is the absorption value of the control (untreated) group; and B refers to the absorption value of the culture medium. IC 50 values (μ g/mL) were calculated with SPSS software. In a competition assay, Prostate cancer cells (5, Experimental protocol. On day 21, after PC-3 cell implantation, mice bearing tumors between 100 and 150 mm 3 were randomized into the following four treatment groups (n = 10): saline controls, MTX treatment, [Dlys 6 ]-LHRH-MTX treatment and MTX plus [Dlys 6 ]-LHRH treatment. Treatments were made in 100 μ L PBS and were given intravenously by tail vein injection twice weekly for 4 weeks. The vehicle-treated group received an equivalent amount of PBS. Tumor volumes were measured with calipers and the body weights of the mice were measured twice a week. Tumor volumes were calculated with the following formula: V(mm 3 ) = length × width 2 /2, where width represents the shortest measurement in millimeters. The animals were euthanized 7 days after the last treatment. Body weights, tumor weights and organ weights were recorded. Statistical analysis. Data were presented as Mean ± SD. Statistical analyses were done by using SPSS 20.0 software. Differences were determined by ANOVA and Student's t test when appropriate. The p value of < 0.05 was considered significant and the p value of < 0.01 was considered highly significant.