Figure 1: JOMD-1307 Inhibits PC Cells growth and progression: (A and B) Graphical representation of the therapeutic effect of JOMD-1307 with increasing concentrations (1 to 20μM) on the viability and proliferation of PC-3 and LNCaP prostate cancer cell line for 72 hrs of incubation, that showed significant inhibition from 1μM. (C and D). PC-3 and LNCaP cells IC50 value represented in log concentration of JOMD- 1307 was approximately 2.0μM. Cell proliferation was presented as the percentage of the number of viable cells to that of the control. Each data point shown is the mean ± SD from n=3. (E) Graphical representation of the effect of JOMD-1307 with increasing concentrations (1 to 20μM) on the PNT1A cell line for 72 hrs of incubation, that showed no significant inhibition before 5μM, with low significant inhibition in increasing concentrations (from 5 to 20μM) compared to PC cell lines.

Figure 2: JOMD-1307 inhibits PC cell division and causes cell death through apoptotic induction: (A- B) The Phospho H-3 method for cell cycle arrest on PC cell lines PC-3 and LNCaP treated with JOMD-1307 in triplicate. Graphical representations showing the various phase ratios of cell cycle arrest when PC-3 and LNCaP cells are treated with the IC50 of the compound for 72 hrs compared with non-treated controls. There was significantly increased cell cycle arrest in the Go-G1-Phase on both PC cell lines. (C-D) The PI Staining method for apoptotic cell death on PC-3 and LNCaP cells was treated with IC50 concentrations of JOMD-1307 in triplicate. Graphical representations revealed significantly increased inducement of apoptotic cells on PC-3 and LNCaP cell lines. (E-F) PC-3 and LNCaP cells were treated with IC50 of JOMD-1307 in triplicate. Graphic representation of immunoblotting (IB) cell lysates showed induction of cleavage PARP and Caspase-8 and -3, AIF, and p53 expression in both PC cell lines. Statistical values represent p < 0.001.

Figure 3: JOMD-1307 increased cellular uptake and heterogeneous distribution in PC cells.

Figure 4: JOMD-1307 decreases Hsp27 expression and interactions and down-regulates the increased transcription in PC cells. (A and B) The inhibitory effect of JOMD-1307 treatment concentrations in a dose-dependent manner (0.5-2.0μM) in triplicate for 72 hrs on PC-3 and LNCaP cells using western blotting analysis to analyze the expression levels of Hsp27 and c-Myc, Graphical representation of results shows Immunoblotting (IB) cell lysates with the respective antibodies the level of inhibitory impact by the JOMD-1307 on the protein expressions. The relative protein quantification was normalized with GAPDH on PC-3 and LNCaP cells. There is significant inhibition in the concentrations (0.5- 2.0μM), but an increased inhibition effect was found at 2.0μM concentration on PC-3 and LNCaP cells. (C) PC-3 and LNCaP cells were treated with JOMD-1307 treatment concentrations (2.0μM). Immunoblotting (IB) lysates and graphical representations of results show significant inhibition of the transcriptional protein HSF-1 expression on the PC cell lines. (D-G) The inhibitory effect of JOMD-1307 treatment concentrations (2.0μM) in triplicate for 72 hrs on PC cell lines (PC-3 and LNCaP cells) using co- immunoprecipitation (co-IP) assay to analyze the interaction levels of Hsp27/c-Myc, and Hsp27/HSF-1 respectively, Graphical representation of results shows Immunoblotting (IB) cell lysates with the respective antibodies the level of inhibitory effect by the JOMD-1307 on the target protein interactions. There was significant disruption in Hsp27/c-Myc and Hsp27/HSF-1 interactions. (H) RT-PCR analysis was used to analyze the level of Hsp27 mRNA expression in PC cell lines. The results revealed a significant decrease of Hsp27 expression normalized with GAPDH compared with the non-treated controls on both LNCaP and PC- 3 cell lines after JOMD-1307 treatment concentration (2.0 μM) for 72 hrs.

Figure 5: JOMD-1307 delays CR prostate tumor growth in preclinical mouse models triggers apoptotic induction, and significantly reduces proliferating tumor cells. An in vivo experiment was conducted with inoculation of androgen-independent PC cells on six-week-old male Bal-C nude mice to form tumor sizes of 90 to 100mm3. The mice were treated with JOMD-1307 and positive control Docetaxel treatment concentration (2mg/kg) twice a week for four weeks on nine (9) mice for each group (JOMD- 1307, Docetaxel, and controls). The statistical measurement of tumor growth was taken twice per week, ex vivo analysis using western blotting for apoptotic protein expression, and the use of Ki67 proliferation testing to determine proliferating cells was analyzed. (A) Statistical representation of tumor growth per week for four weeks after treatment with JOMD-1307 and the control. The results revealed a significant delay of ***P< 0.001 tumor growth in JOMD-1307 treatments compared to the controls. (B) The ex vivo analysis of homogenized xenograft extracts using the western blotting method reveals significant induction of apoptotic cascade (cleaved PARP, cleaved caspase-8 and -3 expressions) by JOMD-1307 treatments compared to the controls. (C) The Ki67 analysis for proliferation testing, using the immunofluorescent (IF) method. The results show a significant reduction of proliferating cells in JOMD-1307 treatments compared to the control.

Figure 6: Schematic representation of JOMD-1307 anticancer therapeutic activities inside PC cell: 1). JOMD-1307 could target the mitochondria, cause cellular stress (ROS production), activate the production of mitochondria AIF and induce apoptotic cascade (cleaved PARP, and Caspase-8 and -3), thereby causing apoptotic PC cell death. 2). JOMD-1307 impairs the c-Myc transcriptional factor to amplify and regulate, and the increased HSF-1 transcription for the upregulation of the expression of Hsp27 stress-induced signaling pathway during cellular stress. 3). The mitochondrial damage caused by JOMD-1307, disrupts the high supply of cellular energy (i.e. ATP) demanded for increased phosphorylation of Hsp27 expression, which leads to a reduced level of phosphorylated Hsp27 (p-Hsp27) in the PC cell, and studies have shown that non-phosphorylated Hsp27 has reduced effect for cellular survival in cancer cells. 4) The reduced level of phosphorylated and non-phosphorylated Hsp27 has none/less effect to actively cytoprotect and interact with the partner proteins, due to the inability of Hsp27 to refold the unfolded/damaged survival proteins from the effect of JOMD-1307, the survival proteins undergo protein degradation. Therefore, upregulates p53 to induce apoptotic PC (CRPC) cell death.

Figure 7: Schematic representation showing JOMD-1307 synthesized with DOTAU nucleolipids (DOU- PEG-2000) as a nano-vehicle for effective transfection and anticancer therapeutic activities to inhibit and delay PC cell viability and tumor growth respectively.