Abstract
Aim: The present study aimed to determine the antitumor efficacy of a new Phenanthroindolizidine alkaloid (PA) derivative, YPC-10157, and to elucidate its mechanism of action. Materials and Methods: The in vitro and in vivo antitumor activity of YPC-10157 was evaluated against several human cancer cell lines and mouse xenograft models, respectively. Cell apoptosis was determined by measuring caspase-3/7 activity. The effect on protein synthesis was assessed using an in vitro cell-free translation assay system. Results: In vitro, YPC-10157 exhibited marked cell growth inhibition and induced apoptosis. In vivo, YPC-10157 had a strong antitumor effect on xenograft models of human colon cancer and leukemia. Moreover, YPC-10157 and its derivatives inhibited protein synthesis and their inhibitory activity on protein synthesis significantly correlated regarding cell growth. Conclusion: YPC-10157 has promising antitumor effects and suggest that its cytotoxic mechanism might involve the inhibition of protein synthesis.
The class of compounds known as phenanthroindolizidine alkaloids (PAs), many of which have been isolated from plants of the Asclepiadaceae family, have been investigated extensively for their anti-inflammatory (1-3) and antitumor (4-24) activity. These compounds were expected to be candidates for anticancer drugs. Tylophorine and its analogs were assessed using a panel of 60 tumor cell lines at the National Cancer Institute and showed strong growth-inhibitory activity toward almost all the cell lines, including P-glycoprotein-overexpressing cells. Moreover, since the spectra of their inhibitory activity were unique among current antitumor compounds, these PAs were expected to become a new class of anticancer agents (4-10). In the 1960s, however, a tylophorine-related compound, tylocrebrine, failed in clinical trials because of side-effects on the central nervous system. Since then, there have been many attempts to improve the physical properties of PAs and to prevent them from passing through the blood–brain barrier (4).Various derivatives of PA derived from natural products have been synthesized and evaluated for antitumor properties in vitro and in vivo. While many of these PA analogs exhibited potent cytotoxic effects against human cancer cell lines in vitro, most of them were inefficacious in vivo, mainly due to poor pharmacokinetic profiles, general toxicity, low water solubility and so on. It has taken a lot of effort to improve their drug-like characteristics to develop them as anticancer drugs. Regarding the mechanism of growth inhibition, it was reported that these compounds inhibited the transcriptional activities of activator protein-1 (AP1) (4), nuclear factor-kappa B (NF-κB) (4-6) and hypoxia inducible factor-1 (HIF1) (7), key transcription factors in cancer cell proliferation, differentiation, resistance to chemotherapy etc. It was also described that they suppressed protein kinase B, also known as AKT, signal activation (5) and induce cell-cycle arrest by modulating cell-cycle regulatory proteins (8-10). Furthermore, it was reported that they interfered with DNA biosynthesis by inhibiting the activity of thymidylate synthase/dihydrofolate reductase (11-13), and could also block protein biosynthesis by affecting the peptide chain elongation step (14-16).We synthesized several derivatives (17-19) of a PA isolated from Ideopsis similis, a butterfly found in Okinawa, Japan (20). Some of these compounds exhibited very strong growth-inhibitory activity against human cancer cell lines in vitro and also showed effective antitumor activity against xenograft tumors in nude mice (17-19). However, their detailed antitumor effects and mechanism of action were unclear. In the present report, we focus on a novel PA derivative, YPC-10157 (Figure 1), and evaluate its cytotoxic effect on several human cancer cell lines and its antitumor effect against human cancer xenograft models. Additionally, to elucidate its mechanism of action, we further investigated its inhibitory effect on protein synthesis as a possible mechanism.
Materials and Methods
Reagents. YPC-10157 and other PA derivatives (Figure 1) were synthesized in our laboratory (18-20). Cycloheximide was purchased from Calbiochem (La Jolla, CA, USA). For in vitro assays, all compounds were dissolved in dimethylsulfoxide (DMSO) as a stock solution and the final concentration of DMSO was less than 0.1%.
Cell culture. All human cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Human lung cancer cell line A549, colon cancer HT-29 and HCT116 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA). Breast cancer MCF7, HCC1806, MDA-MB-231 and MDA-MB-468, promyelocytic leukemia HL-60 were maintained in RPMI medium 1640 (Invitrogen). Each medium was supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen), and cells were grown at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
Cell viability assay. Cell viability was assayed in a 96-well plate using a TetraColor ONE (Seikagaku Corp., Tokyo, Japan), according to the manufacturer's protocol. Briefly, exponentially growing cells were seeded at a density of 1×103 cells/well. The next day, serially diluted compounds were added to each well. After 96 h of incubation, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt, WST-8, was added to each well and the plates were incubated at 37°C for 1 h. Absorbance was then measured at 450 nm using a SPECTRA Max PLUS384 (Molecular Devices, Sunnyvale, CA, USA). The half maximal (50%) inhibitory concentration (IC50) for cell viability was defined as the concentration of compound that inhibited cell viability by 50% compared to solvent-treated control cells.
Caspase-3/7 activity assay. Caspase-3/7 activities in the cells were measured with Caspase-Glo 3/7 Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Cells were seeded in a 96-well plate at 5×103 cells/well, and then treated with YPC-10157 for 24 h. Treated cells were incubated with 10 μl of the assay reagent at room temperature. After 1 h, luminescence was measured to calculate caspase-3/7 activities.
In vivo tumor xenograft model. Six-week-old male BALB/c nude mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). Cells were suspended in saline (2×107 cells/ml) and 0.1 ml of the cell suspension was injected subcutaneously into the right flank of each mouse. For the HCT-116 xenograft model, when tumors reached a mean volume of 160 to 260 mm3 (day 1), mice were randomly divided into three groups of five mice per group; YPC-10157 mesylate was dissolved in 5% glucose and administered intravenously at 22.2 mg/kg or 44.4 mg/kg on days 1, 3, 5, 8, 10, 12, 15, and 17. For the HL-60 xenograft model, when tumors reached a mean volume of 90 to 240 mm3 (day 1), mice were divided into three groups of four mice per group; YPC-10157 mesylate was administered intravenously at 16.7 mg/kg or 33.3 mg/kg on days 2, 6, and 10.
Tumor size was measured using calipers twice a week and the body weight of each mouse was monitored for assessing toxicity. Tumor volume was estimated by using the formula length×width2×1/2. On day 22 for the HCT116 model, and day 16 for the HL60 model, the antitumor activity was evaluated by weighing the tumor tissues. The tumor growth inhibition rate, IR (%), was calculated as [1−(average tumor weight of each treated group)/(average tumor weight of control group)]×100.
All experiments with animals were conducted in accordance with the Guidelines of the Yakult Central Institute for Microbiological Research and protocols approved by The Institutional Animal Care and Use Committee of the Yakult Central Institute (Approved No. 10-0224).
Cell-free translation assay. A cell-free translation assay was carried out using the TNTR Coupled Reticulocyte Lysate System (Promega), according to the manufacturer's directions. Briefly, firefly luciferase cDNA as a template and compounds were added directly to the TNTR Lysate System and incubated for 1.5 h at 30°C. Firefly luciferase activity was measured using the Steady-GloR Luciferase assay system (Promega) with SPECTRA Max M5e (Molecular Devices). The IC50 for protein synthesis was defined as the concentration of compound that inhibited protein synthesis by 50% compared to the solvent-treated control.
Statistical analysis. For comparisons of more than two groups, the data were analyzed for statistical significance with Dunnett's multiple comparison test. For the correlation analysis, the correlation coefficient was calculated by the linear least-squares regression method. Probability values of less than 0.05 were considered significant.
Results
Inhibitory effect of YPC-10157 on growth of human cancer cell lines in vitro. The effect of YPC-10157 on cell proliferation after 96 h treatment was examined in eight human cancer cell lines: A549, HT-29, HCT116, MCF7, HCC1806, MDA-MB-231, MDA-MB-468 and HL-60. As shown in Table I, YPC-10157 inhibited the growth of all cell lines in the lower nanomolar range. Out of all the cell lines used in this study, HL-60 showed the highest sensitivity to YPC-10157, with an IC50 of 0.419 nM.
Activation of apoptotic pathway by YPC-10157. Caspases are the main enzymes that mediate apoptosis. To determine whether YPC-10157 inhibits the growth of cancer cells via the apoptotic pathway, we measured changes in the caspase activity in HL-60 cells after 96 h treatment. As shown in Figure 2, YPC-10157 treatment dose-dependently increased caspase-3 and -7 activities.
Antitumor effect of YPC-10157 on tumor xenografts in vivo. We further evaluated the antitumor activity of YPC-10157 in human cancer cell xenografts using HCT116 and HL-60 which showed higher sensitivity to YPC-10157 in vitro (Table I). In the HCT116 model, treatment with YPC-10157 mesylate at 22.2 or 44.4 mg/kg significantly inhibited the growth of tumors, with IR values of 49.4% and 64.6%, respectively, compared to the vehicle-only on day 22 (Figure 3A). Although the administration of YPC-10157 at 44.4 mg/kg led to 15% body weight loss compared with day 1, the body weight recovered after the treatment ceased. In the HL-60 model, treatment with YPC-10157 at 16.7 or 33.3 mg/kg more markedly inhibited the growth of tumors, with IR values of 57.4% and 96.8%, respectively, compared to the vehicle on day 16 (Figure 3B). In particular, YPC-10157 at 33.3 mg/kg reduced tumor volume on day 13 compared to day 1. No body weight loss was observed during the treatment of animals of this model.
Effect of YPC-10157 on protein synthesis. Next, we investigated the mechanism of the growth-inhibitory effect. As previously mentioned, it seemed tylophorine and its analogs have several mechanisms of action (4-16). Because it was also reported that similar structural analogs had different potency or selectivity under each mechanism of action (21), how YPC-10157 inhibited growth was obscure. Therefore, we investigated its effect on protein synthesis as a possible mechanism of its cytotoxic action. To elucidate whether YPC-10157 directly affects protein synthesis, we performed an in vitro cell-free translation assay using rabbit reticulocyte lysate (22). YPC-10157 exhibited a dose-dependent inhibition of protein synthesis as effectively as cycloheximide, a protein synthesis inhibitor (Figure 4).
Correlation between the cell-growth inhibition and protein synthesis inhibition of PA derivatives. To further confirm the inhibitory activity of YPC-10157 on protein synthesis associated with its cytotoxic action, we examined the inhibitory activities of several YPC-10157-based PA derivatives (Figure 1) on the protein synthesis and cell growth of A549 and HT-29 cells. All the derivatives, including YPC-10157, inhibited both the growth of cancer cell lines and protein synthesis (Table II), but their inhibitory potencies differed. Therefore, to examine the correlation between the cell growth inhibition and protein synthesis inhibition, IC50 values for cell growth inhibition of all derivatives were plotted against those for protein synthesis inhibition (Figure 5). As a result, protein synthesis inhibition was significantly correlated with growth inhibition in both A549 cells (Figure 5A; r=0.95, p<0.001) and in HT-29 cells (Figure 5B; r=0.92, p<0.001).
Discussion
YPC-10157 is a novel PA derivative optimized for anti-proliferative activity and water solubility. YPC-10157 showed potent growth-inhibitory activity against all tested human cell lines derived from lung cancer, colon cancer, breast cancer and leukemia, through an apoptotic mechanism, in vitro (Table I, Figure 2). These results suggest that YPC-10157 may have therapeutic utilities against a broad spectrum of cancer types.
Some reports have indicated that a hydroxyl moiety on C14 of the phenanthroindolizidine structure is important for strong antitumor activity in vivo (4, 23) and that the methoxy moiety on the phenanthrene ring may contribute to less cytotoxicity in vitro (24) and metabolic instability in vivo (23). In the case of YPC-10157, although it lacks a hydroxyl moiety at C14 and it has two methoxy moieties on its phenanthrene ring, it exhibited a strong antitumor effect with mild toxicity in vivo (Figure 3). Our previous study had also shown that YPC-10157 has more potent antitumor activity and lower lethal toxicity than its hydroxylated derivatives (17). These results indicate that YPC-10157 has more favorable drug-like properties than previously reported PA derivatives.
In the cell-free translation assay, YPC-10157 dose-dependently inhibited protein synthesis (Figure 4). In the correlative evaluation, YPC-10157 and its derivatives inhibited both cancer cell growth and protein synthesis (Table II), and these activities correlated significantly with each other (Figure 5). Previous reports suggested that PA and its derivatives have various mechanisms of action (4-16). A recent report described that phenanthrene-based analogs, being structurally fairly similar, exhibit different mechanisms of action (21). Our study suggests that these PA derivatives, including YPC-10157, exhibit antitumor activity by inhibiting protein synthesis. It has been reported that tylophorines inhibit protein synthesis by blocking chain elongation (14). It seems likely YPC-10157 and its derivatives have a similar mechanism of action, but further study is needed. In conclusion, a novel PA derivative, YPC-10157, has strong anticancer activity in vitro and in vivo, and its mechanism of action might be through disruption of protein synthesis. These results indicate that YPC-10157 may be a potential candidate for anticancer therapy.
Acknowledgements
We appreciate the technical assistance of Ms. Yukiko Nishiyama.
Footnotes
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This article is freely accessible online.
- Received March 20, 2014.
- Revision received May 21, 2014.
- Accepted May 22, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved