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Research Articles: Therapeutics, Targets, and Development
P276-00, a novel cyclin-dependent inhibitor induces G1-G2 arrest, shows antitumor activity on cisplatin-resistant cells and significant in vivo efficacy in tumor models
Departments of 1 Pharmacology and 2 Medicinal Chemistry, Nicholas Piramal Research Centre, Nicholas Piramal India Ltd., Mumbai, India and 3 Oncotest GmbH, Freiburg, Germany
Requests for reprints: Kalpana S. Joshi, Department of Pharmacology, Nicholas Piramal Research Center, Nicholas Piramal India Ltd., 1-Nirlon Complex, Goregaon (East), Mumbai 400 063, India. Phone: 91-22-3081-8421; Fax: 91-22-3081-8411. E-mail: kjoshi{at}nicholaspiramal.co.in
Abstract
P276-00, a flavone that inhibits cyclin-dependent kinases, has been identified by us recently as a novel antineoplastic agent. In this study, we have selected a panel of human tumor cell lines and xenografts to allow determination of selectivity and efficacy of P276-00. When tested against a panel of 16 cisplatin-sensitive and cisplatin-resistant cell lines, the antiproliferative potential of P276-00 was found to be 
30-fold higher than cisplatin. Studies to show tumor sensitivity using clonogenic assay in 22 human xenografts indicated that P276-00 was 26-fold more potent than cisplatin, and further, it was also found to be active against cisplatin-resistant tumors of central nervous system, melanoma, prostate, and renal cancers. Further, we studied the effects of P276-00 on cell cycle progression by flow cytometry using asynchronous and synchronous population of tumor and normal cells. Asynchronous population of human prostate carcinoma (PC-3) and human promyelocytic leukemia (HL-60) cells when exposed to P276-00 showed arrest of slow-growing PC-3 cells in G2-M with no significant apoptosis observed up to 72 h. Unlike PC-3, significant apoptosis was seen in fast-growing HL-60 cells at 6 h. However, synchronized human non–small cell lung carcinoma (H-460) and human normal lung fibroblast (WI-38) cells showed arrest of cells in G1. H-460 cells undergo apoptosis, which increases with longer exposure to the compound and also after exposure to P276-00 for 48 h followed by recovery. In contrast, the normal cells (WI-38) remain arrested in G1 with no significant apoptosis up to 72 h of exposure and also after 48 h of P276-00 treatment followed by recovery, confirming our previous results that P276-00 was less effective against normal cells compared with cancer cells. After promising in vitro results, P276-00 was checked for in vivo efficacy in murine tumor and human xenograft models. Growth inhibition of murine colon cancer (CA-51) was significant when P276-00 was administered i.p. at 50 mg/kg daily for 20 treatments. However, in murine lung carcinoma model (Lewis lung), an increased dose of 60 mg/kg (30 mg/kg twice daily) administered every alternate day i.p. for seven treatments showed significant inhibition in the growth. Further studies were undertaken to establish the efficacy profile of P276-00 in human tumor xenograft models. In the two xenograft models studied, P276-00 showed potent in vivo antitumor potential. Compound P276-00 at a dose of 35 mg/kg administered daily via the i.p. route for 10 days showed significant (P < 0.05) inhibition in the growth of human colon carcinoma HCT-116 xenograft. Furthermore, P276-00 at a dose of 50 mg/kg once daily and 30 mg/kg twice daily administered via i.p. route for 20 treatments significantly (P < 0.05) inhibited growth of human non–small cell lung carcinoma H-460 xenograft. Thus, the in vitro cellular potency, together with in vivo antitumor activity, confirms the potential of P276-00, a cyclin-dependent kinase inhibitor as an anticancer molecule. [Mol Cancer Ther 2007;6(3):926–34]
Introduction
The G1-S cell cycle checkpoint controls the passage of eukaryotic cells from G1 phase into the DNA synthesis S phase. This process is dependent on the activities of cyclin-dependent kinases (Cdk) that are sequentially regulated by cyclins D, E, and A (1–4). Cyclin D associates with Cdk4/Cdk6 and the catalytic activities of the assembled holoenzymes are first manifested by mid-G1, increase to a maximum at the G1-S transition, and contribute to G1 exit (5–8). Cdk2 associates with either cyclin E or cyclin A, and the resultant kinase activities increase at the G1-S transition or in the early S phase, respectively. Cdk4/6-cyclin D, Cdk2-cyclin E, and the transcription complex that includes pRb and E2F are pivotal in controlling progression through the late G1 restriction point (9). The phosphorylation of pRb late in G1, initially triggered by Cdk4/6 and later accelerated by Cdk2, induces pRb to dissociate from E2F. This event can transactivate S-phase genes encoding for proteins that amplify the G1-S phase switch and are required for DNA replication (10–14). We have identified P276-00, a potent Cdk4-D1 and Cdk1-B inhibitor, which showed significant antiproliferative effects against various human cancer cell lines in nanomolar range (earlier publication). It was also found to be highly selective for cancer cells compared with normal cells (earlier publication). In the present study, P276-00 was found to be highly active against cisplatin-resistant cell lines. We have shown that P276-00 can inhibit tumor cell growth by interrupting cell cycle progression either in G1 or in G2, accompanied by apoptosis. In contrast, normal cells remain arrested in G1 and do not undergo apoptosis. More importantly, P276-00 showed excellent in vivo activity against murine and human tumor models of colon and lung carcinoma without toxic side effects. These findings suggest that P276-00 is a candidate for further preclinical and clinical development, as well as a model for the synthesis of other flavonoids that might potently delay cell cycle progression leading to apoptosis.
Materials and Methods
Cell Lines
The human cancer cell lines used in the cell proliferation assay by Prof. Fiebig laboratory (Oncotest, Freiburg, Germany) were maintained at 37°C in a humidified atmosphere (95% air, 5% CO2) in RPMI 1640 (Invitrogen, Karlsruhe, Germany) supplemented with 10% FCS (Sigma, Deisenhofen, Germany) and 0.1 mg/mL gentamicin (Invitrogen).
Cells were routinely passaged once or twice weekly. They are maintained no longer than 20 passages in culture. Cell lines were established from solid human tumor xenografts as described by Roth et al. (15). The origin of the xenografts was described by Fiebig et al. (16). The cell lines DLD1, HCT-116, HT-29, H-460, MCF-7, and PC3M were kindly provided by the National Cancer Institute (Bethesda, MD).
Clonogenic and Propidium Iodide Assay
The clonogenic and propidium iodide (PI) assays were carried out at Oncotest. A modified PI assay was used to assess the effects of the platin compounds on the growth of the human tumor cell lines (17). Briefly, cells were harvested from exponential phase cultures by trypsinization, counted, and plated in 96 well flat-bottomed microtiter plates at a cell density dependent on the cell line (5–12,000 viable cells per well).
After 24-h recovery to allow the cells to resume exponential growth, 20 µL culture medium (six control wells per plate) or culture medium containing the test compound was added to the wells. Each concentration was plated in triplicate. Compounds were applied at five concentrations with one log increments (0.003, 0.03, 0.3, 3, and 30 µg/mL). Following 4 days of continuous drug exposure, cell culture medium with or without drug was replaced by 200 µL of an aqueous PI solution (7 µg/mL). Because PI only passes leaky or lysed cell membranes, DNA of dead cells will be stained and measured, whereas living cells will not be stained. To measure the proportion of living cells, cells were permeabilized by freezing the plates, resulting in death of all cells. After thawing of the plates, fluorescence was measured using the Cytofluor 4000 microplate reader (excitation, 530 nm; emission, 620 nm), giving a direct relationship to the total cell number. Growth inhibition/stimulation was expressed as treated/control x 100 (%T/C). Antitumor activity was defined as inhibition of tumor growth to <30% to the medium-treated control cells. The coefficient of variation [SD / mean x 100 (%)] was in nearly all experiments <20%. IC50 and IC70 values were determined by plotting compound concentration versus cell viability.
The clonogenic assay uses solid human tumor xenografts, which are mechanically disaggregated and subsequently incubated with an enzyme cocktail consisting of 41 units/mL collagenase, 175 units/mL DNase, and 100 units/mL hyaluronidase in RPMI 1640 at 37°C for 30 min. The cells were washed twice and passed through sieves of 200 and 50 µm mesh size. The percentage of viable cell was determined using trypan blue exclusion.
The clonogenic assay was done as published previously (18). A modified PI assay was used to assess the effects of the compounds on the growth of the human tumor cell lines.
Analysis of Cell Cycle Distribution
The human non–small cell lung carcinoma (H-460), human prostate cancer (PC-3), human promyelocytic leukemia (HL-60), and the normal lung fibroblast (WI-38) cell lines were seeded in 25 mm3 tissue culture flask at a density of 0.5 x 106 cells per flask. After 24 h, cells were treated with about thrice IC50 of P276-00 for cancer cells (i.e., 1.5 µmol/L for 0, 6, 12, 24, and 48 h). For cell recovery studies, H-460 and WI-38 cells were treated with 1.5 µmol/L P276-00 for 48 h followed by medium without compound for 0, 6,18, 24, and 48 h of recovery. Both detached and adherent cells were harvested at different time points. After washing in PBS, cells were fixed in ice-cold 70% ethanol and stored at –20°C. Cells were washed twice with PBS to remove fixative and resuspended in PBS containing 50 µg/mL PI and 50 µg/mL RNaseA. After incubation at room temperature for 20 min, cells were analyzed using flow cytometry.
Flow Cytometry
A Becton Dickinson (San Jose, CA) FACSCalibur flow cytometer was used for these studies in accordance with the manufacturer's recommendations. The argon ion laser set at 488 nm was used as an excitation source. Cells with DNA content between 2N and 4N were designated as being in G1, S, and G2-M phases of the cell cycle, as defined by the level of red fluorescence. Cells exhibiting <2N DNA content were designated as sub-G1 cells. The number of cells in each cell cycle compartment was expressed as a percentage of the total number of cells present.
Murine Tumor Models
Murine colon (CA-51) and Lewis lung tumors were excised from preexisting tumors in animals, minced on ice, and passed through a cell strainer. The cell suspension was then centrifuged at 1,000 rpm. Cell pellet was resuspended in saline (0.85% normal) to give a count of 4.2 million/0.2 mL suspension and placed on ice. BALB/c mice were injected with 0.2 mL of the cell suspension s.c. on the right flank and observed daily for tumor appearance. For colon carcinoma, 24 h later (day 1), the animals were randomized into two groups, control (group I) and 50 mg/kg P276-00 (group II). P276-00 solution in water (50 mg/kg) was administered every day i.p. to animals in group II for 20 days. Animals in control group (group I) were administered water i.p.
For mouse Lewis lung model, when the tumors attained a diameter of 5 mm, they were randomized into three groups, control (group I), 35 mg/kg P276-00 administered i.p. (group II), and 60 mg/kg P276-00 administered i.p. (group III). P276-00 solution in water was administered at 35 mg/kg every day i.p. for 14 days in group II and at 30 mg/kg twice daily (60 mg/kg/d) in group III every alternate day until seven treatments. Animals in control group were administered water i.p.
Animals in both test models were observed every day for signs of health deterioration and animal weight was recorded daily. Tumor diameters were measured using a digital Vernier caliper, when they attained an average diameter of 5 mm and followed by every 2 to 6 days.
Tumor weight in milligram was calculated using the formula for a prolate ellipsoid:
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as 3.
Treated to control ratio (T/C%) on a given day was calculated using the formula:
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Growth inhibition (GI) was calculated as GI on day X = 100 – T/C% on day X.
Tumor Xenograft Model
The human colon cancer (HCT-116) and non–small cell carcinoma (H-460) cells were grown in RPMI 1640 containing 10% fetal bovine serum and harvested. Cells were resuspended in saline at 3.2 and 6.4 million cells/0.2 mL volume, respectively, and placed on ice. Severe combined immunodeficient mice were injected with 0.2 mL of the cell suspension s.c. on the right flank and observed daily for tumor appearance. When the tumors attained a diameter of 5 mm, they were randomized into two groups. For HCT-116 tumor model, control (group I), water was administered every day i.p. for 10 days and, 35 mg/kg P276-00 (group II), P276-00 solution in water was administered every day i.p. also for 10 days.
For H-460 tumor xenograft, the severe combined immunodeficient mice were randomized in three groups, control (group I), 50 mg/kg P276-00 administered once daily i.p. for 20 days (group II), and 30 mg/kg P276-00 administered twice daily i.p. for a total of 18 treatments (group III). Animals in control group were administered water.
Animals in both the test models were observed every day for signs of health deterioration and animal weight was recorded daily. Tumor weight in milligram and growth inhibition were calculated as described in tumor murine model.
Results
P276-00 Shows Potent Antiproliferative Activity against Cisplatin-Resistant Tumors
The effect of P276-00 as well as cisplatin, a standard cytotoxic chemotherapeutic drug used for the treatment of various cancers, was studied on a panel of 16 human tumor cell lines in a total of 48 experiments (three experiments per cell line; Table 1
). All cell lines were treated with different concentrations of P276-00 and cisplatin for 48 h. The main evaluation criteria were antitumor activity (mean IC50 and IC70), tumor selectivity, and its activity compared with cisplatin. The results of these experiments suggested that P276-00 had a cytotoxic effect across a range of tumor types. P276-00 showed very good anticancer activity in vitro, with a mean IC70 value of 0.50 µg/mL (1.1 µmol/L), which was clearly more active than cisplatin, which showed a IC70 of 11.9 µg/mL (40 µmol/L) in 16 tumor cell lines tested. P276-00 was 30 times more potent than cisplatin and showed very distinct selectivity for certain tumor cells. The most sensitive cell lines were three of four colon carcinomas (DLD1, HCT-116, and CXF 94L), the lung cancer cell line LXFA 629L, both mammary cancer cell lines MCF-7 and MAXF 401NL, as well as MEXF 462NL derived from a melanoma. P276-00 was also active on certain cisplatin-resistant tumor cell lines of colon cancer DLD1 and CXF 94L and the mammary cancer cell line MCF-7.
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Regulatory molecules governing early cell cycle progression, such as cyclin D1/Cdk4/pRb, are frequent targets of genetic alterations in cancer (21). As such, the cell cycle regulatory pathway may represent a useful target for drug and gene therapy approaches. The flavone P276-00 has been evaluated here for antitumor activity in vitro and in vivo. In vitro evaluation was done in 16 tumor cell lines using PI assay and in 22 human tumor xenograft-derived panel using a clonogenic assay. Its antiproliferative activity was compared with cisplatin, a standard chemotherapeutic drug used in almost all advanced cancers. Cisplatin exerts its antitumor effects via the formation of DNA adducts and cross-links. Cisplatin-induced DNA damage results in cell cycle arrest, primarily at the S and G2 checkpoints, providing the opportunity for DNA damage repair before mitosis (22, 23). Unrepairable DNA damage often results in activation of the apoptotic pathway. In 16 human tumor cell lines and 22 human tumor xenografts, P276-00 was found to have a highly potent antiproliferative effect with a mean IC50 of 0.550 and 0.620 µmol/L, respectively. It was found to be more potent than cisplatin and showed significant antitumor activity with 30 times more potent (IC70) than cisplatin. P276-00 showed very distinct selectivity for certain colon, lung, mammary, and melanoma tumor cells and was more active on certain cisplatin-resistant tumor cell lines of colon and mammary. The fact that cisplatin-resistant cell lines were sensitive to P276-00 indicates its potential use in the clinic against cisplatin-resistant tumors. These results encouraged us to investigate whether P276-00 induces a synergistic effect with cisplatin and other conventional chemotherapeutic agents in various cancers and these studies are ongoing. The compound used in the present study was further evaluated in clonogenic assay because this assay better reflects the in vivo clinical situation than in vitro cytotoxicity assays that use permanent tumor cell lines. In addition, it has been found to be the most predictive test for further in vivo evaluation of anticancer drugs in the clinic (24). In this assay, among all the xenografts tested, compound P276-00 exhibited pronounced antitumor activity against tumor lines derived from central nervous system, melanoma, prostate, kidney (RXF), and sarcoma. The data also indicated that P276-00 was clearly more potent and showed a slightly different antitumor selectivity compared with cisplatin.
Based on the role of Cdk4 and Cdk1 in cell cycle progression, an inhibitor of these two enzymes would be predicted to produce a G1 or a G2 arrest. Consistent with this expectation, asynchronous population of cells treated with P276-00 blocked the cells in both G1 and G2. Furthermore, cells synchronized in the G0 phase by serum starvation maintained a G1 block. Prolonged exposure to the compound resulted in apoptosis in cancer cells, whereas in the normal fibroblast cell line, no apoptosis was seen even after prolong exposure, thus confirming our previous observation that P276-00 spares normal fibroblast cells. The normal and cancer cell line when subjected to recovery after compound treatment indicated that cancer cell lines continue to undergo apoptosis, which is quite significant compared with the normal cell line where the increase in apoptosis is negligible. Therefore, the selective killing effect of P276-00 against cells that are actively proliferating could be exploited in developing this compound as a potential antitumorigenic therapeutic agent (25).
The murine tumor and the human xenograft models showed the efficacy of P276-00 in colon and lung carcinoma when given by the i.p. route and with different schedule of administration. In the murine tumor model, a reduction in colon tumor growth by 81% was observed using i.p. administration when given at a dose of 50 mg/kg daily for 20 treatments. This also indicates that when a higher dose is administered once daily (i.e., 50 mg/kg; Fig. 3), it is as effective as a similar dose (60 mg/kg; Fig. 4) given as 30 mg/kg twice daily every alternate day. Similarly, the xenografts of human colon and lung models both showed very good efficacy when treated with P276-00. Therefore, P276-00 was as efficacious in the colon carcinoma model at a lower dose and less frequent regimen (i.e., almost half that for lung carcinoma model). This could be due to the differences in the intrinsic sensitivity of the tumor cells to P276-00. Although, we have not yet attempted to ascertain the mechanism of cell death in the xenograft tumor models, it remains possible that a delay in cell cycle progression accompanied by apoptosis may account for some of the observed reduction in tumor growth rate. However, further studies with P276-00 alone and in combination with other conventional drugs in other tumor models in vitro and in vivo seem warranted to enhance our understanding and accelerate the application of this promising chemotherapeutic agent.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10/ 4/06; revised 12/ 5/06; accepted 2/ 1/07.
References
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