PST Analogs have Selective Anti-Cancer Activity Greater Than Standard Chemotherapeutics & Natural PST

The preclinical advancement of PST has been hindered by its low yield from its natural source and complexities in its chemical synthesis. Previously, we have shown modest to comparable anti-cancer activity of a 7-deoxyPST analog in comparison to natural PST25,26,27. Recently, we have synthesized several PST analogs with a C-7 hydroxyl group (Fig. 1), thus, possessing the complete pharmacophore attributed to the anticancer activity of PST and related alkaloids24. A comprehensive screen of anti-cancer activity of these analogs, in parallel with 7-deoxyPST analogs, PST, and common chemotherapeutics was completed on a battery of cancer cell lines as well as non-cancerous cells using the WST-1 colorimetric assay (Fig. 2). Taken as a whole, SVTH-7, followed by SVTH-6 and SVTH-5, had the most potent activity, with SVTH-7 having much greater activity than natural PST while SVTH-6 and -5 possessed comparable or greater efficacy than natural PST with regards to their half-maximal inhibitory concentration (IC 50 ) values (Table 1). As predicted, SVTH-6 and SVTH-5, which are C-7 hydoxylated forms of JCTH-4 and JCTH-3, respectively, were markedly more effective than their 7-deoxyPST counterparts.

Figure 1 Structure of PST and PST Analogs. Full size image

Figure 2: PST Analogs have Selective Anti-Cancer Activity Greater Than Standard Chemotherapeutics & Natural PST. Cancer and non-cancerous cells were treated with PST analogs, PST, Taxol, Doxorubicin (DOX), Gemcitabine (GEM), and Cisplatin for 48 hours. With the WST-1 assay, absorbance was read at 450 nm and expressed as a percent of control (DMSO). Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. X-axis: Concentration (μM) (using a Log 10 Scale). Y-axis: Absorbance at 450 nm (% of Control). Full size image

Table 1 IC 50 Values of PST, PST Analogs, and Standard Chemotherapeutics. Full size table

Triple negative breast cancer (TNBC) lacks the estrogen, progesterone, and HER2 receptor, and thus traditional breast cancer therapies, including hormone therapy and Herceptin, are not effective28. Standard chemotherapeutics for TNBC include Taxol and Doxorubicin (DOX). Interestingly, SVTH-7, -6, and -5 had lower IC 50 values than Taxol and DOX in the TNBC cell lines MDA-MB-231 and MDA-MB-468. SVTH-7 and SVTH-6 were also more effective than Gemcitabine (GEM), the standard chemotherapeutic for notoriously chemoresistant pancreatic cancer29, in the BxPC-3 and PANC-1 pancreatic cancer cell lines. Furthermore, JCTH-3 and -4, and SVTH-5, -6, and -7 were more potent than Cisplatin and GEM, having lower IC 50 values in the NCI-H23 non-small cell lung cancer cell line, a commonly chemoresistant cancer. Moreover, SVTH-7 and -6 had lower IC 50 values than Taxol in MV-4-11 leukemia, and U-87 MG glioblastoma. Similar results were observed with MCF7 breast adenocarcinoma, OVCAR-3 ovarian adenocarcinoma, and Hep G2 hepatoma cells (Supplemental Fig. 1a). Notably, the IC 50 values of PST and its analogs in the AG09309 and CCD-18Co non-cancerous cells were well above those observed in the cancer cells lines tested, demonstrating a selective therapeutic window. Additional time points, doses and statistical analyses of compounds tested are shown in Supplemental Fig. 1b–w.

PST Analogs Induce Apoptosis Selectively in Cancer Cells

To evaluate cell death caused by PST analogs, the Annexin V binding assay and propidium iodide (PI) staining was done in parallel to monitor early apoptosis30 and necrotic or late apoptotic cell death, respectively31. PST analogs were effective in inducing apoptosis in the U-937, E6-1, and MV-4-11 lymphoma and leukemia cell lines as well as BxPC-3 pancreatic adenocarcinoma cells (Fig. 3a). SVTH-7, followed by SVTH-6 and SVTH-5, was the most effective in inducing apoptosis compared to natural PST and the 7-deoxyPST analogs. Staurosporine (STS) was used as a positive control for apoptotic induction32. Interestingly, SVTH-6 and -7 were more efficacious in inducing apoptosis in BxPC-3 cells compared to Gemcitabine (GEM), the standard chemotherapeutic for pancreatic cancer. Non-cancerous cells, including peripheral blood mononuclear cells from healthy volunteers 1 (PBMCs V1) and 2 (PBMCs V2), AG09309 normal human fibroblasts, and NCM460 normal human epithelial cells were much less sensitive to apoptotic induction. Only SVTH-7 and SVTH-6, at doses substantially higher than what is required to induce apoptosis in cancer cells, demonstrated mild toxicity in some of these non-cancerous cells (Fig. 3b). Cell death analyses of additional non-cancerous peripheral blood mononuclear cells from other healthy volunteers with similar resilience against PST analog treatment are depicted in Supplemental Fig. 2a. Furthermore, to see if apoptosis could occur in actively dividing PBMCs V1, concanavalin a (Con A), a known inducer of proliferation of peripheral blood mononuclear cells was added. Although Con A was able to induce proliferation, PST and PST analogs were still not able to trigger apoptosis in PBMCs V1, which is in contrast with Taxol treatment, indicating that PST and its analogs do not target cells because they are actively dividing (Supplemental Fig. 2b). HEK-293 human embryonic kidney cells were also unresponsive to PST and PST analog treatment (Supplemental Fig. 2c). Representative micrographs of E6-1 leukemia cells undergoing apoptosis after 48 hours of PST analog treatment are shown in Fig. 3c. SVTH-7, -6, and -5 were the most effective at yielding condensed cell morphology, nuclear condensation, and Annexin V (green) and PI (red) fluorescence, which are indicative of apoptotic induction.

Figure 3: PST Analogs Induce Apoptosis Selectively in Cancer Cells. Annexin V binding and PI staining of cells treated for 48 hours was monitored with image-based cytometry. (a) Cancer cell lines. (b) Non-cancerous cells. Values are expressed as mean ± SD from at least 3 independent experiments. *p < 0.01 vs. DMSO control. (c) Annexin V binding (green), PI staining (red), and Hoechst (blue) monitored with microscopy. Cell morphology is shown using differential interference contrast (DIC) microscopy. Scale bar = 25 μm. Micrographs are representative of 3 independent experiments. Full size image

PST Analogs Activate the Intrinsic Pathway of Apoptosis in Cancer Cells

Mitochondria play a pivotal role in the induction of intrinsic apoptosis. When dysfunctional, these organelles can permeabilize and release apoptogenic factors, leading to the execution of apoptosis33. One such factor is cytochrome c (Cyto c), which upon its release from the mitochondria, leads to the conversion of Pro-Caspase-9 (Pro-Casp-9) to Caspase-9 (Casp-9), which in turn cleaves Pro-Caspase-3 (Pro-Casp-3) to Caspase-3 (Casp-3)34. The executioner caspase, Casp-3, exerts its lethal effects by cleaving a multitude of cellular proteins needed for cellular function, structural stability, and survival35. MV-4-11 leukemia cells were treated with PST and PST analogs and no noticeable activation of caspases were observed at 3 hours. At 6 hours, there is prominent activation of Casp-9 and -3 with SVTH-7 and to a lesser extent with SVTH-6. After 12 hours, JCTH-4, SVTH-5, -6, and -7 treatment yielded prominent cleavage of Pro-Casp-9 and Pro-Casp-3, as well as DNA damage, as indicated by the presence of γ-H2AX, in MV-4-11 cells (Fig. 4a), demonstrating their ability to induce the aforementioned caspase-dependent pathway of apoptosis. Densitometric analyses are depicted in Supplemental Fig. 3a. Moreover, SVTH-7 caused the release of Cyto c from the mitochondria of MV-4-11 cells (Supplemental Fig. 3b). Although at 12 hours there is activation of Caspase-8, leukemia cells that are dominant negative for the Fas-Associated Death Domain (FADD) (DN FADD Jurkat), a critical component of the extrinsic pathway and activation of Caspase-8, were still very sensitive to PST and PST analogs compared to corresponding leukemia cells with functional FADD (Supplemental Fig. 3c). This indicates that the extrinsic pathway of apoptosis is not a predominant pathway responsible for PST and PST-analog-induced cytotoxicity. Likewise, there was no observable differences in the conversion of LC3-I to LC3-II, a marker of autophagy36, with PST and PST analog treatment, suggesting autophagy to possess little role in the anti-cancer activity of these compounds (Fig. 4a and Supplemental Fig. 3b).

Figure 4: PST Analogs Activate the Intrinsic Pathway of Apoptosis in Cancer Cells. (a) Western blot analysis of cell lysates of MV-4-11 Leukemia cells treated with PST, PST Analogs, Taxol, and staurosporine (STS) for 3, 6 and 12 hours. TMRM was used to monitor MMP in (b) MV-4-11 leukemia cells, (c) non-cancerous peripheral blood mononuclear cells from volunteer 1 (PBMCs V1), and normal human fibroblasts (AG09309) with image-based cytometry. *p < 0.01 vs. DMSO control. (d) TMRM fluorescence microscopy counterstained with Hoechst (cyan). Scale bar = 25 μm. All images are representative of at least 3 independent experiments. Full size image

Complimenting these findings, permeabilization of mitochondria, as seen with dissipation of mitochondrial membrane potential (MMP), was first noticeable at 3 hours in MV-4-11 cells with SVTH-7 treatment, and at 6 hours with JCTH-4 and SVTH-6, with a more pronounced effect at 12 hours as shown by a decrease of TMRM red fluorescence (Fig. 4b). Additional time points and doses are shown in (Supplemental Fig. 3d) and similar results are observed with E6-1 leukemia and BxPC-3 pancreatic adenocarcinoma cells (Supplemental Fig. 3e and f). This effect on mitochondria was observed to be selective towards cancer cells as PMBCs V1 and AG09309 had very minimal or no observable decreases in MMP with PST and PST analog treatment (Fig. 4c). Representative micrographs depicting TMRM fluorescence of MV-4-11 cells treated with PST and PST analog, Taxol, and staurosporine (STS) are shown in Fig. 4d. Collectively, these findings suggest that PST analogs are able to act on the mitochondria to induce the intrinsic pathway of apoptosis.

PST and PST-Induced Apoptosis is Highly Dependent on Mitochondrial Membrane Permeabilization and Partially Dependent on Caspase Activity

Following MMP collapse and mitochondrial permeabilization, apoptogenic factors are released and cause subsequent activation of caspases. To study the dependence of caspases in PST analog-induced apoptosis, the Z-VAD-FMK broad-spectrum caspase inhibitor was used (Fig. 5a). Interestingly, this inhibitor was able to prevent activation of Casp-3 by SVTH-6 and -7 in MV-4-11 (Fig. 5b) and E6-1 (Fig. 5c) leukemia cells. Densitometric analyses are given in Supplemental Fig. 4a and b. This was able to partially rescue E6-1 and MV-4-11 (Supplemental Fig. 4c) leukemia cells from PST and PST analog-induced apoptosis, suggesting both caspases and other apoptosis inducers are involved in such cell death. Doxorubicin (DOX) was used as a positive control for Z-VAD-FMK-mediated rescue37.

Figure 5: PST and PST-Induced Apoptosis is Highly Dependent on Mitochondrial Membrane Permeabilization and Partially Dependent on Caspase Activity. (a) E6-1 cells were pre-treated with Z-VAD-FMK caspase inhibitor for 1 hour and then treated with PST analogs and DOX. Annexin V binding and PI staining was quantified with image based cytometry. *p < 0.01 vs. DMSO control (comparison of viable cells only); #p < 0.001 vs. respective groups untreated with Z-VAD-FMK (comparison of viable cells only). (b) Western blot analysis was performed on MV-4-11 cells pre-treated with Z-VAD-FMK for one hour and treated with 1 μM DOX, 0.5 μM SVTH-6, and 0.01 μM SVTH-7 for an additional 12 hours. (c) Jurkat cells (E6-1 leukemia cells) were pre-treated with Z-VAD-FMK for 1 hour or DMSO. These cells, along with Jurkat cells over-expressing the anti-apoptotic protein Bcl-2 (++Bcl-2), were then treated with 1 μM DOX, 0.5 μM SVTH-6, and 0.1 μM SVTH-7 for 24 hours. Western blot analysis was performed on corresponding cell lysates. (d) Annexin V binding and PI staining, as well as (e) TMRM was quantified with image based cytometry on these Jurkat after 48 hours of treatment. *p < 0.01 vs. DMSO control (comparison of viable cells only); $p < 0.01 vs. DMSO control of ++Bcl-2 Jurkat; #p < 0.001 vs. respective groups without over-expression of Bcl-2; @p < 0.01 vs. ++Bcl-2 Jurkat treated with 0.5 μM SVTH-5 alone. All quantitative values are expressed as mean ± SD from at least 3 independent experiments. Western blots are representative of at least 3 independent experiments. Full size image

Jurkat cells over-expressing the anti-apoptotic protein Bcl-2 (++Bcl-2), a protein known to stabilize mitochondria and prevent mitochondrial membrane permeabilization, were then treated with PST analogs and Doxorubicin (DOX) for 24 hours. ++Bcl-2 Jurkat cells had drastically lower levels of Casp-3 and -9 activation and γ-H2AX compared to Jurkat cells with no over-expression of Bcl-2 (E6-1) (Fig. 5c). Densitometric analyses are given in Supplemental Fig. 4b Furthermore, ++Bcl-2 Jurkat cells were drastically less susceptible to PST analog-induced apoptosis (Fig. 5d) and experienced significantly less MMP dissipation (Fig. 5e) compared to Jurkat without this over-expression of this mitochondrial stabilizing protein. Interestingly, the Bcl-2 inhibitor EM20-25, which disrupts interactions between Bcl-2 and Bax38, was able to sensitize the ++Bcl-2 Jurkat to the PST analog SVTH-5 and potentiated its ability to induce apoptosis and dissipate MMP in these cells (Fig. 5d and e). Therefore, this body of work suggests that PST analog-induced apoptosis is highly dependent on mitochondrial membrane permeabilization.

PST Analogs Act on Cancer Cell Mitochondria and Cause Mitochondrial Dysfunction

One of the first events of mitochondrial dysfunction is the generation of reactive oxygen species (ROS)39. Using H 2 DCFDA, an indicator of ROS, PST analogs and PST were shown to increase the production of ROS in MV-4-11 leukemia and U-937 lymphoma cells after 3 hours of treatment (Fig. 6a). Piperlongumine (PL) and paraquat (PQ) were used as a positive control for ROS generation40,41.

Figure 6: PST Analogs Act on Cancer Cell Mitochondria and Cause Mitochondrial Dysfunction. (a) H2DCFDA was used to measure whole cell ROS in MV-4-11 and U-937 cells treated for 3 hours with image-based cytometry. *p < 0.01 vs. DMSO control. (b) The MitoXpress® Xtra - Oxygen Consumption Assay was used to monitor oxygen consumption via fluorescence generation. Cells were treated, and the fluorescent MitoXpress® reagent was added and monitored at Ex. 380 nm and Em. 650, every 2 minutes for 2 hours at 37 °C. Oxygen consumption rates were calculated by measuring the slopes of the linear regions of the oxygen consumption curves. *p < 0.001 vs. DMSO control. (c) Western blot analysis of E6-1 following treatment with the indicated drugs for 6 hours. Results are representative of 3 independent trials. (d) Detection of ATP levels following treatment with SVTH-5, -6, and -7, PST, and Taxol using the luciferase-luciferin ATP determination assay. Amount of ATP was expressed as number of moles of ATP over micrograms of protein. Results are shown as the mean ± SD from at least 3 independent experiments. *p < 0.05 vs DMSO control. (e) Western Blot analysis of Cyto c release (of post mitochondrial supernatant) from directly treated mitochondria isolated from MV-4-11 cells for 2 hours. SDHA was probed in the mitochondrial pellet samples as loading controls. All quantitative values are expressed as mean ± SD from at least 3 independent experiments. Western blots are representative of at least 3 independent experiments. Full size image

Oxygen consumption of cells is a direct indicator of mitochondrial function42. To assess the effect of PST analogs on oxygen consumption, the MitoXpress® Xtra - Oxygen Consumption Assay was used (Fig. 6b). SVTH-6, -7, and PST were able to effectively decrease the rate of oxygen consumption in E6-1 leukemia cells. In U-937 lymphoma cells, SVTH-5, -6, and -7 were effective in decreasing oxygen consumption rates. Antimycin A (AMA), a complex III inhibitor of the ETC, was used as a positive control for oxygen consumption cessation. Phosphorylation of AMPK (p-AMPK), a marker for activating several cell survival pathways, and total amount of ATP were measured following 6 and 12 hours of treatment and there was an increase in p-AMPK and a decrease in total amount of ATP in each group compared to the control, with the exception to SVTH-5 (Fig. 6c and d). These results indicate that PST analogs are effective in reducing oxygen consumption and therefore, mitochondrial function.

To determine if PST analogs are able to directly act on cancer cell mitochondria to release Cyto c, mitochondria isolated from MV-4-11 cells were directly treated with PST analogs for 2 hours and the release of Cyto c was monitored (Fig. 6e). Interestingly, such treatment caused the release of this apoptogenic factor with the most pronounced effect observed with SVTH-6 and -7. Therefore, together these findings demonstrate that PST and PST analogs act on cancer cell mitochondria and cause mitochondrial dysfunction directly.

PST Analog-Induced Apoptosis is Dependent on Functional Complex II and III of the Mitochondrial Electron Transport Chain

As we have shown PST analogs to act on cancer cell mitochondria and affect their functioning, we investigated the role of mitochondrial ETC complexes in PST analog-induced apoptosis using the complex II inhibitor Thenoyltrifluoroacetone (TTFA) and the complex III inhibitor AMA43,44. Interestingly, TTFA was able to rescue these cells from SVTH-7 insult, making the percentage of dead cells statistically similar to those of the DMSO control following 48 hours of treatment, preventing Casp-3 activation and reducing the levels of γ-H2AX (Fig. 7a,b,d). Cell salvation by TTFA was specific to SVTH-7 as this inhibitor had no significant effect on Taxol, DOX and STS treatment. A slightly less dramatic rescue was observed with AMA (Fig. 7a,c,d). TTFA and AMA were also able to prevent SVTH-7-induced Casp-9 activation (Supplemental Fig. 5a).

Figure 7: PST Analog-Induced Apoptosis is Dependent on Functional Complex II and III of the Mitochondrial Electron Transport Chain. MV-4-11, and U-937 cancer cells were pre-treated with TTFA and AMA for 1 hour and then treated with PST analog, staurosporine (STS), Doxorubicin (DOX), and Taxol for 48 hours. Annexin V binding (green) and PI staining (PI) (red) was observed with (a) microscopy and (b,c) quantified with image based cytometry. Scale bar = 25 μm. *p < 0.01 vs. DMSO control (comparison of viable cells only); #p < 0.001 vs. respective groups without TTFA or AMA (comparison of viable cells only). (d) Western blot analysis of AMA and TTFA pre-treated cells treated with 1 μM DOX, and 0.01 μM SVTH-7 with MV-4-11 cells and 0.025 μM SVTH-7 with U-937 cells. Images are representative of 3 independent experiments. Full size image

In addition, TTFA was able to protect cancer cell mitochondria from SVTH-7-induced dissipation of MMP and bring the percentage of TMRM positive cells to levels that are similar to values observed in the DMSO control treated group which can be seen at both 12 (Supplemental Fig. 6) and 48 hours (Fig. 8a and c). A similar but less dramatic rescue of MMP was observed with AMA (Supplemental Fig. 6 and Fig. 8b,c). Prevention of mitochondrial membrane permeabilization by TTFA was specific to SVTH-7 treatment as no significant changes in the percentage of TMRM positive cells was observed with Taxol, DOX, and STS treatment in conjunction with this inhibitor. Interestingly, inhibition of complex I with the inhibitor Rotenone (ROT)44 and uncoupling the ETC from ATP production with FCCP had no significant effect of SVTH-7 activity (Supplemental Figs 7 and 8). The functionality of the aforementioned ETC modulators on mitochondrial function was validated by an oxygen consumption assay as seen in Supplemental Fig. 9. Therefore, these observations imply that functional complex II, and, to a lesser extent, complex III are required for SVTH-7 to exert its pro-apoptotic effects in cancer cells.

Figure 8: PST Analog-Induced MMP Dissipation is Dependent on Functional Complex II and III of the Mitochondrial Electron Transport Chain. MV-4-11 and U-937 cancer cells were pre-treated with (a) TTFA or (b) AMA for 1 hour and then treated with PST analogs, staurosporine (STS), Doxorubicin (DOX), and Taxol for 48 hours. TMRM fluorescence was quantified with image-based cytometry. *p < 0.01 vs. DMSO control; #p < 0.001 vs. respective groups without TTFA or AMA. All values are expressed as mean ± SD from at least 3 independent experiments. (c) Representative fluorescent micrographs of 3 independent experiments of U-937 lymphoma cells stained with TMRM (red) and Hoechst (blue). Scale bar = 25 μm. Full size image

PST Analogs Selectively Induce Apoptosis in 3D Spheroid Models of Cancer

The three-dimensional architecture of tumors has been shown to dictate the responsiveness of cancer cells to chemotherapeutics45. To evaluate the efficacy of PST analogs in a more architecturally accurate context, cells were grown in three-dimensional spheroid culture on basement membrane extract (BMX) coated surfaces for 48 hours, which provides a scaffold for cells to form three-dimensional structures46, and treated with PST analogs for 72 hours. SVTH-7, -6, -5 and natural PST were the most effective on the HCT 116 colorectal cancer and BxPC-3 pancreatic cancer spheroids as determined by the WST-1 viability assay (Fig. 9a). Interestingly, SVTH-6 and natural PST had comparable anti-cancer activity compared to GEM, the current standard chemotherapeutic for pancreatic cancer29, in BxPC-3 spheroids while SVTH-7 had significantly superior activity compared to GEM.

Figure 9: PST Analogs Selectively Induce Apoptosis in 3D Spheroid Models of Cancer. Cells were cultured on basement membrane extract (BMX) to form 3D spheroids, grown for 48 hours, and treated for 72 hours. (a) The WST-1 reagent was used to quantify viability. Absorbance was read at 450 nm and expressed as a percentage of control. Values are expressed as mean ± SD from triplicates of at least three independent experiments. *p < 0.05; **p < 0.005; ***p < 0.0005 vs. DMSO control. #p < 0.001 vs. 0.5 μM GEM. (b) Confocal microscopy was used to monitor Annexin V binding (green) and (c) TMRM fluorescence (red). Cells were counterstained with NucRed Live 647 ReadyProbes® Reagent to visualize nuclei (blue in B, cyan in C). Scale bar = 20 μm. Micrographs are representative of 3 independent experiments. Full size image

Annexin V binding was monitored in HCT 116 colorectal cancer spheroids (Fig. 9b). Similarly to the STS positive control for apoptosis, HCT 116 cells treated with SVTH-6 and -7 were positive for Annexin V binding, indicated by the green fluorescence. This was accompanied by nuclear condensation and cell shrinkage, as depicted in fluorescence and corresponding DIC micrographs respectively, which are all indicative of apoptosis. Minimal Annexin V binding was present in the DMSO solvent control treated group. In the DIC micrograph of the solvent control, spheroids were dramatically larger and cells exhibited large, round, healthy cellular morphology. NCM460 normal colon mucosa spheroid cells were dramatically less sensitive to SVTH-6 and -7. Unlike the STS positive control, minimal Annexin V binding was observed in both the solvent control and SVTH-6 and -7 treated cells, which exhibited healthy nuclear and cell morphology.

MMP collapse was monitored as another marker of apoptosis (Fig. 9c)47. SVTH-6 and -7 were able to dissipate MMP in HCT 116 and BxPC-3 cancer cells in spheroid culture as indicated by the dissipation of red TMRM fluorescence. However, no such dissipation was evident in the NCM460 normal colon mucosa spheroid cells. Together, these results indicate that PST analogs SVTH-6 and -7 are both effective and selective against cancer cells grown in three-dimensional spheroid culture.

PST Analogs Decrease Growth of Tumors in Xenograft Mouse Models

To evaluate the anti-cancer activity of PST analogs in vivo, cancer cells were subcutaneously injected into the flanks of immunocompromised mice. After palpable tumors were established approximately 1 week after injections, mice were treated with 3 mg/kg of PST analogs intratumorally 3 times a week for approximately 5 weeks. JCTH-4 and SVTH-5 were able to reduce the growth of both HCT 116 and HT-29 colorectal tumor xenografts with SVTH-5 showing greater efficacy (Fig. 10a and b). SVTH-6 was also effective in reducing growth of HT-29 tumors (Fig. 10c). Furthermore, SVTH-6 and -7 were very effective in reducing the growth of HCT 116 colorectal cancer and U-87 MG glioblastoma tumor xenongrafts as tumor volumes were drastically smaller than the DMSO solvent control treated tumors (Fig. 10c). Mice treated with JCTH-4, SVTH-5, -6, and -7 all increased in mass throughout the studies and did not significantly differ from the masses of mice treated with DMSO solvent control (Fig. 10a–c). These findings demonstrate that PST analogs are able to decrease the growth of tumors in vivo and are well tolerated by mice at their effective doses.