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ZSTK474 is a novel orally applicable phosphoinositide 3-kinase-specific inhibitor that strongly inhibits cancer cell proliferation. To further explore the antitumor effect of ZSTK474 for future clinical usage, we studied its combined effects with radiation. The proliferation of HeLa cells was inhibited by treatment with X-rays alone or ZSTK474 alone. Combination treatment using X-rays then ZSTK474 given orally for 8 days, starting 24 h post-irradiation, significantly enhanced cell growth inhibition. The combined effect was also observed for clonogenic survival with continuous ZSTK474 treatment. Western blot analysis showed enhanced phosphorylation of Akt and GSK-3β by X-irradiation, whereas phosphorylation was inhibited by ZSTK474 treatment alone. Treatment with ZSTK474 after X-irradiation also inhibited phosphorylation, and remarkably inhibited xenograft tumor growth. Combined treatment with X-rays and ZSTK474 has greater therapeutic potential than radiation or drug therapy alone, both in vitro and in vivo. (Cancer Sci 2011; 102: 1176–1180)
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinase that phosphorylates the 3-hydroxyl group of the inositol ring of phosphoinositides.(1) As PI3Ks have been shown to be important targets in cancer therapy, development of PI3K inhibitors has attracted attention from both academic and industrial researchers. Recently, several novel PI3K inhibitors have been developed and some of them are now undergoing clinical trial.(2)
Among the novel PI3K inhibitors, ZSTK474 has unique properties.(1,3–6) ZSTK474 is a pan-PI3K inhibitor: it inhibits all four PI3K isoforms in an ATP-competitive manner. Among all of the PI3K isoforms, PI3Kδ was inhibited most potently by ZSTK474. PI3K-related kinases are a group of protein kinases with a catalytic core structure similar to PI3K, but they lack the lipid kinase activity. This group includes mTOR, DNA-PK, ATM, and ATR proteins, of which the latter three are known to be involved in DNA damage-related responses. The inhibition activity of ZSTK474 against DNA-PK and mTOR was determined, and was shown to be far weaker compared with that observed against PI3K. These results indicate that ZSTK474 was the most specific agent among these PI3K inhibitors.(1,4,6) The inhibition selectivity of ZSTK474 for PI3K over DNA-PK was significantly higher than other PI3K inhibitors such as NVP-BEZ235, PI-103, and LY294002.(6)In vitro, ZSTK474 induced marked G0/G1 arrest in various human cancer cells without any obvious apoptosis.(3,5) This pan-PI3K inhibitor, given orally, showed potent in vivo antitumor efficacy on cancer xenografts at both early and advanced stages, without obvious toxicity being observed.(3)
ZSTK474 strongly inhibited tumor growth. Thus, to consider the clinical usage of ZSTK474, its combination with another therapy is a practical choice. In single drug therapy, the use of multiple agents, such as chemotherapeutic drugs and radiation, increases effectiveness and potency in clinical cancer therapy. Radiation can be used as an agent to increase the anticancer effect of ZSTK474, as ZSTK474 effectively inhibits the downstream of the PI3K pathway.
In general, the combination of chemotherapy and radiotherapy can be classified into three categories based on the time sequence of the treatments: neoadjuvant (chemotherapy before radiotherapy); concurrent; and adjuvant (chemotherapy after radiotherapy) chemo-radiotherapy. The time sequence of the treatments usually affects the results. It is known that the sensitivity of cells to radiation depends on the cell cycle phases.(7) The cells are most sensitive to low linear energy transfer (LET) ionizing radiation when irradiated at G2/M phase and are most resistant when irradiated at late S phase. Other phases, such as G1 and early S, show intermediate sensitivity. As ZSTK474 induces arrest in the G0/G1 phase of the cell cycle, where radiation sensitivity is not the highest, ZSTK474 was not used in this study in the typical manner of radiation sensitizers. In addition, ZSTK474 is a specific PI3K inhibitor, and it showed weaker inhibitory activity against DNA-PKcs, which is thought to be essential for the double strand break (DSB) repair process after radiation exposure.
We then examined the time sequence where ZSTK474 was applied not immediately, but much later after irradiation. In this study, significant combination effects of X-rays and ZSTK474 were observed both in vitro and in vivo.
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Ionizing radiation activates the PI3K/Akt pathway,(9,10) so the PI3K survival pathway can be a major target for combined treatments. Many reports have shown the radiosensitizing effect of low molecule PI3K inhibitors in vitro.(11,12) However, the inhibitors extensively studied so far, such as wortmannin and LY294002, lack specificity and showed unacceptable toxicity in animal studies.(13) Recently, a new generation of PI3K inhibitors, including ZSTK474, has been developed and their in vivo anticancer effect has been closely examined.(3,14–18) Furthermore, their combination with other agents might be desirable for more adequate usage of the drugs in future clinical therapy. In the present study, we have clearly showed that the combination of X-rays and ZSTK474 (treated 24 h after X-rays) remarkably inhibited the proliferation of human cancer cells (HeLa) and the growth of the xenograft tumor. Only 100 mg/kg ZSTK474 was required to almost completely inhibit tumor growth in vivo if the tumor was X-irradiated once before treatment. A higher concentration (400 mg/kg) was required for a similar effect with ZSTK474 alone.(3)
Treatment of non-small-cell lung cancer with a combination of PI3K inhibitor and γ-irradiation in vivo was reported recently.(19) In the report, dual PI3K/mTOR blockade by BEZ235, a novel, orally applicable pan-PI3K inhibitor, was shown to effectively sensitize non-small-cell lung cancer to the pro-apoptotic effects of ionizing radiation both in vivo and in vitro, where BEZ235 was treated before irradiation to enhance the radiation effect. As the specificity of BEZ235 to PI3K is not high compared to ZSTK474 and considerable inhibition of DNA-PK is expected,(6) the radiosensitizing effect of BEZ235 might be partly explained by the inhibition of DNA-PK, by which the DNA repair process should be disturbed.(20) In the present study, however, we used ZSTK474, a novel, and orally applicable PI3K-specific inhibitor after X-ray treatment. The timing of ZSTK474 application was 24 h after irradiation, so the DNA repair process related to DNA-PK should be completed.(20) Therefore, inhibition of DNA-PK by ZSTK474, even if such activity is present, is not likely to be the major mechanism for the significant combination effect observed in the present study.
A strong combination effect was observed for in vitro cell proliferation assay when the cells were treated for a longer time with ZSTK474. The colony-forming assay also showed that continuous treatment, rather than transient treatment (24 h), significantly reduced the surviving fraction after X-irradiation. This reduction in the surviving fraction might be due to the strong inhibition of cell proliferation by ZSTK474, especially after treatment with X-rays, rather than the induction of actual cell death. This interpretation is supported by colony sizes, where ZSTK474 treatment alone remarkably reduced the colony sizes, but the effect was stronger for cells irradiated first then treated with the drug. ZSTK474 alone does not induce apoptosis.(3,5) A significant difference in the number of apoptotic cells was not observed in the tumor between the samples treated with X-rays alone and with the combination of X-rays and ZSTK474 (data not shown).
ZSTK474 alone significantly inhibited phosphorylation of Akt and its downstream molecule GSK-3β. In contrast, X-irradiation enhanced the phosphorylation of Akt and GSK-3β. The activation of PI3K/Akt signaling is associated with the radio-resistance of many cancer cells.(21–23) When ZSTK474 was applied after X-irradiation, the enhancement of the activation of Akt and GSK-3β was reversed and the reduced phosphorylation of these proteins was observed. It is postulated that the activation of the PI3K pathway in irradiated tumors is an important survival strategy for these cells. However, to understand the molecular mechanism(s) of the therapeutic efficacy of this combination, its effects on other survival or death factors should be examined. Therefore, treatment with ZSTK474 after X-irradiation leading to the inhibition of PI3K pathway, which the irradiated cells try to activate, might be related to effective tumor growth inhibition, observed by the combination treatment in this study.
In this study, we clearly showed that the combination of ZSTK474 with X-rays (continuous application of ZSTK474 after X-irradiation) results in dramatic tumor growth inhibition in vitro and in vivo. Although the precise molecular mechanism to explain these results remains to be elucidated, this phenomenon might be worth exploring for clinical application. An alternative sequence, X-irradiation after ZSTK474, should be examined next. Furthermore, combination with heavy-ion radiotherapy, an effective cancer therapy modality for certain tumors, is also a fascinating theme.