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

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 × 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 × 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 × 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:assuming specific gravity of tumor as 1 and π as 3.

Treated to control ratio (T/C%) on a given day was calculated using the formula:

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.

For further characterization of P276-00, its effect against tumor cells was studied in a clonogenic assay. In vitro clonogenic assays have been widely used for assessing anticancer drug effects and for preclinical screening of new anticancer agents. Promising reports of good correlation between results of in vitro soft agar colony-forming assays and patient's clinical response or resistance to establish antineoplastic agents have been published (18–20). The effect of P276-00 and cisplatin on the growth of tumor cells to colonies was examined in 22 human xenografts of various tumor types. P276-00 and cisplatin were tested in concentrations ranging from 0.001 to 100 μmol/L, using continuous exposure. Antitumor activity of the compound was defined as inhibition of colony formation in treated groups by >70% in relation to an untreated control group. The compound P276-00 significantly inhibited the formation of colonies in a dose-dependent manner. Antitumor activity of P276-00 was clearly more pronounced compared with cisplatin (Tables 2 and 3 ). These results also indicate the selectivity of P276-00 against certain tumor types. The most sensitive tumor models for P276-00 were the tumors of central nervous system (CNXF SF268), non–small cell lung cancer (LXFE 397), mammary cancer (MAXF 401), melanoma (MEXF 276 and MEXF 462), prostate cancer (PRXF 22RV1), renal cancer (RXF 393 and RXF 944LX), and the sarcoma (SXF 1301; Table 2). Less sensitive tumors were seen among colon cancers (CXF 1103 and CXF 243), the adeno lung cancers (LXFA 297), and prostate cancers (PRXF DU145 and PRXF PC3MX). Antitumor selectivity of the reference compound cisplatin resulted in a slightly different selectivity pattern. The most sensitive tumor models were mammary cancer (MAXF 401 and MAXF MX1) and melanoma (MEXF 514). Resistant tumor models for treatment with cisplatin were the adeno lung cancer (LXFA 297), melanoma (MEXF 276), and the prostate cancer (PRXF DU145; Table 2). Inhibition in sensitive tumor models was seen in a concentration range between 1 and 10 μmol/L for P276-00, whereas cisplatin showed antitumor activity in a concentration range from 10 to 100 μmol/L (Table 3). At concentration of 1 μmol/L, P276-00 was active in 41% (9 of 22) of the tested tumor models, at 10 μmol/L, 91% (20 of 22) of the tumor models were inhibited in colony formation, and at 100 μmol/L, P276-00 showed antitumor activity in all tumor models tested (22 of 22). The results for cisplatin showed significantly less antitumor efficacy in comparison with P276-00 at all the concentrations tested. There was no inhibition of colonies at 1 μmol/L, 9% (2 of 22) of the tumor models at 10 μmol/L, and inhibition of 64% (14 of 22) of the tumor models at 100 μmol/L (Table 3).

Effect of P276-00 on Cell Cycle Progression

The possible mechanism by which P276-00 exerts cytotoxic effect on tumor cells was investigated using flow cytometry. The cell cycle distribution analysis was carried out using asynchronous and synchronous populations. Initial experiments were conducted with asynchronous population of human prostate cancer cells (PC-3) and promyelocytic leukemia cells (HL-60). For PC-3 cells, the fraction of cells with G₂-M DNA content increased reciprocally from 19% in control to 41% in the 72-h P276-00 (1.5 μmol/L)–treated sample with percentage apoptosis of 4.28% at the end of 72 h. The G₁ or G₀ fraction marginally decreased form 57% to 47% (Fig. 1A ). In contrast to PC-3, a slow-growing cell line, an asynchronous population of the fast-growing cell line HL-60 when treated with P276-00 showed significant apoptosis from 6 h onwards. HL-60 cells treated with 1.5 μmol/L P276-00 showed >75% apoptosis at the end of 24 h and no detectable cells were present in G₁ and G₂. This indicates that P276-00 has more pronounced effect on fast-growing cancer cells (Fig. 1B). For a more precise examination of where in the cell cycle P276-00 may be acting to inhibit cell cycle progression, non–small cell lung carcinoma cells (H-460) and human normal lung fibroblast (WI-38) were released from serum starvation–induced cell cycle arrest at G₀ into medium containing either no drug or 1.5 μmol/L P276-00. From 6 h onwards, after release from serum starvation, the fraction of H-460 cells with G₁ DNA content increased progressively from 77.42% to 82.77% by 24 h. At 24 h, apoptosis of 2.89% was observed, which is indicated by the sub-G₁ fraction. These cells when incubated further up to 72 h showed an increase in the cell fraction undergoing apoptosis, from 27.42% at 48 h to 34.78% at 72 h (Table 4 ). Therefore, when the cells are blocked at G₀ by serum starvation, P276-00 can prevent entry of the cells into the S phase. We compared the effect of P276-00 on human normal lung fibroblast (WI-38). The treated cells remain arrested in G₁ with the fraction of these cells remaining constant between 80% to 84%, whereas the cells in the control population moved from the G₁-S to G₂-M and back to G₁ phase by 72 h (Table 4). These cells undergo negligible apoptosis of only 3% to 4% by 72 h compared with the cancer cells. In another study, synchronized normal lung fibroblast cell line WI-38 and the non–small cell lung cancer cell line H-460 were subjected to recovery after treatment with 1.5 μmol/L P276-00 for 48 h. The cells were allowed to grow in medium without the compound for 6, 18, 24, and 48 h for recovery. After recovery at different time points, including 0 h, the cells were harvested and cell cycle analysis was done. For normal cell line WI-38, the apoptosis after 48 h was not significant (5.47%) compared with H-460 (27.69%), which is approximately five times more compared with WI-38 (Fig. 2A and B ). These observations confirm our previous results that P276-00 is less potent for normal slow-growing cells compared with cancer cells.

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Figure 1.

A, P276-00 causes G₂-M arrest with apoptosis in asynchronous population of PC-3 when exposed to 1.5 and 5 μmol/L P276-00 for various time points. The DNA histograms were generated by flow cytometry and the percentage of cells in each phase of the cell cycle was determined using ModFit analysis. B, P276-00 causes significant apoptosis in asynchronous population of HL-60 cells from 6 h onwards when treated with concentrations of 1.5 and 5 μmol/L P276-00 for various time points.

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Figure 2.

A, cell cycle progression during recovery at 6, 18, 24, and 48 h of non–small cell lung carcinoma (H-460) cells treated with 1.5 μmol/L P276-00 for 48 h. B, cell cycle progression during recovery at 6, 18, 24, and 48 h of normal lung fibroblast cell line (WI-38) treated with 1.5 μmol/L P276-00 for 48 h.

In vivo Efficacy Studies

Dose-finding studies (data not shown) with P276-00 identified a dose of 78 mg/kg/d administered i.p. once daily for 5 days, as maximum tolerated dose. Antitumor efficacy of P276-00 was first evaluated in murine tumor models of colon (CA-51) and Lewis lung carcinoma. For CA-51, following tumor establishment and randomization of tumor-bearing animals into groups, P276-00 was administered at a dose of 50 mg/kg daily via the i.p. route for 20 days. A significant in vivo antitumor activity of P276-00 in CA-51 murine model was seen. Average tumor weight in control animals on day 33 was 563.1 ± 174.1 mg, whereas average tumor weight in treated group was 172.5 ± 69.5 mg. We observed a significant growth inhibition of 81% at day 33. Relative tumor volume of the P276-00–treated animals was significantly lower than that of the vehicle control on measurement on days 20, 24, 27, 29, 31, and 33. The T/C ratio (ratio of median treated tumor weight T to median control tumor weight C, expressed as percent) was 19% at the end of the experiment (Fig. 3 ). For Lewis lung carcinoma model, P276-00 was administered at a dose of 35 mg/kg daily i.p. for 14 days and at a dose of 30 mg/kg twice daily (60 mg/kg/d) every alternate day for 7 days. The percentage growth inhibition with the two different dosing regimens on day 17 was 43% and 70%, respectively. Thus, these results showed that P276-00, when given twice daily every alternate day (60 mg/kg/d) for 7 days, was more efficacious (Fig. 4 ). The statistical analysis of the tumor weights on the last day of tumor measurement by the Student's t test (for two-sample, unequal variance with a one-tail distribution) shows that the weight differences are statistically significant with a P value <0. 05. In both murine tumor models, no significant reduction in weight loss was seen.

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Figure 3.

Average tumor growth of murine colon carcinoma (CA-51) over a period of 33 d after randomization. P276-00 was administered i.p. for 20 d. Control animals received i.p. injection of water.

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Figure 4.

Average tumor growth of murine lung cancer over period of 16 d after randomization.

We further evaluated the antitumor efficacy of P276-00 in xenograft models of human colon carcinoma (HCT-116) and non–small cell lung carcinoma (H-460). I.p. administration of P276-00 at 35 mg/kg daily for 10 days showed significant (P < 0.05) reduction in HCT-116 tumor growth and average weight loss of 10% compared with that observed in the vehicle-treated group. The average tumor weight on day 21 for control and P276-00 was 1,349 ± 130.9 and 517.2 ± 70.3 mg, respectively. The calculated percentage of growth inhibition on day 21 after randomization was >70% (Fig. 5 ).

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Figure 5.

Average tumor growth of human colon carcinoma (HCT-116) xenograft over a period of 15 d. P276-00 at 35 mg/kg was administered i.p. from days 1 to 10. Control animals received i.p. injections of water.

Similarly, P276-00 was administered i.p. at 50 mg/kg once daily for 20 days and 30 mg/kg twice daily for 20 days in mice bearing H-460 tumors. The average tumor weight on day 40 postinjection in control animals was 5,782.4 ± 1,321.4 mg, whereas the average tumor weights in the treated groups were 1,444.3 ± 446.3 and 1,877.6 ± 436.5, respectively. Both schedules and dosing regimens were equally efficacious with percentage growth inhibition of tumor of 75.7% and 68.4%, respectively, on day 40 (Fig. 6 ). After chronic treatment, animals had an average weight loss of 12% to 15%. In both the xenograft models, the statistical analysis of the tumor weights on the last day of tumor measurement by the Student's t test shows that the differences are statistically significant with a P value <0.05.

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Figure 6.

Average tumor growth of non–small cell lung carcinoma (H-460) over a period of 40 d. P276-00 was administered once daily at 50 mg/kg i.p. and twice daily at 30 mg/kg via the i.p. route. Control animals received i.p. water for injections.

Discussion

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.