Induction of MYCN-amplified neuroblastoma differentiation through NMYC suppression using PPAR-γ antagonist.

Neuroblastomas are the most common extracranial solid tumors in children and have a unique feature of neuronal differentiation. Peroxisome proliferator-activated receptor (PPAR)-γ is reported to have neuroprotective effects in addition to having antitumor effects in various cancers. Thus, we aimed to clarify the role of PPAR-γ agonist and antagonist in malignant neuroblastomas, which also possess neuronal features. In MYCN-amplified neuroblastoma CHP212 cells, treatment with the PPAR-γ antagonist GW9662 induced growth inhibition in a dose-dependent manner. In addition, the PPAR-γ antagonist treatment changed cell morphology with increasing expression of the neuronal differentiation marker tubulin beta 3 (TUBB3) and induced G1 phase arrest and apoptosis in MYCN-amplified neuroblastoma. Notably, the PPAR-γ antagonist treatment significantly decreased expression of NMYC, B-cell lymphoma 2 (BCL2) and bromodomain-containing protein 4 (BRD4). It is implied that BRD4, NMYC, BCL2 suppression by the PPAR-γ antagonist resulted in cell growth inhibition, differentiation, and apoptosis induction. In our in vivo study, the PPAR-γ antagonist treatment induced CHP212 cells differentiation and resultant tumor growth inhibition. Our results provide a deeper understanding of the mechanisms of tumor cell differentiation and suggest that PPAR-γ antagonist is a new therapeutic and prevention option for neuroblastomas.

Salicylic acid directly binds to ribosomal protein S3 and suppresses CDK4 expression in colorectal cancer cells.

Colorectal cancer is a significant cause of morbidity and represents a serious public health issue in many countries. The development of a breakthrough preventive method for colorectal cancer is urgently needed. Aspirin has recently been attracting attention as a cancer preventive drug, and its inhibitory effects on the development of various cancers have been reported in several large prospective studies. However, the underlying molecular mechanisms have not yet been elucidated in detail. In the present study, we attempted to identify the target proteins of aspirin using a chemical biology technique with salicylic acid, the main metabolite of aspirin. We generated salicylic acid-presenting FG beads and purified salicylic acid-binding proteins from human colorectal cancer HT-29 cells. The results obtained showed the potential of ribosomal protein S3 (RPS3) as one of the target proteins of salicylic acid. The depletion of RPS3 by siRNA reduced CDK4 expression and induced G1 phase arrest in human colorectal cancer cells. These results were consistent with the effects induced by the treatment with sodium salicylate, suggesting that salicylic acid negatively regulates the function of RPS3. Collectively, the present results show the potential of RPS3 as a novel target for salicylic acid in the protective effects of aspirin against colorectal cancer, thereby supporting RPS3 as a target molecule for cancer prevention.

Stabilization of CDK6 by ribosomal protein uS7, a target protein of the natural product fucoxanthinol.

Cyclins and cyclin-dependent kinases (CDKs) regulate the cell cycle, which is important for cell proliferation and development. Cyclins bind to and activate CDKs, which then drive the cell cycle. The expression of cyclins periodically changes throughout the cell cycle, while that of CDKs remains constant. To elucidate the mechanisms underlying the constant expression of CDKs, we search for compounds that alter their expression and discover that the natural product fucoxanthinol downregulates CDK2, 4, and 6 expression. We then develop a method to immobilize a compound with a hydroxyl group onto FG beads® and identify human ribosomal protein uS7 (also known as ribosomal protein S5) as the major fucoxanthinol-binding protein using the beads and mass spectrometry. The knockdown of uS7 induces G1 cell cycle arrest with the downregulation of CDK6 in colon cancer cells. CDK6, but not CDK2 or CDK4, is degraded by the depletion of uS7, and we furthermore find that uS7 directly binds to CDK6. Fucoxanthinol decreases uS7 at the protein level in colon cancer cells. By identifying the binding proteins of a natural product, the present study reveals that ribosomal protein uS7 may contribute to the constant expression of CDK6 via a direct interaction.

Citrus limonL.-Derived Nanovesicles Show an Inhibitory Effect on Cell Growth in p53-Inactivated Colorectal Cancer Cells via the Macropinocytosis Pathway.

Edible plant-derived nanovesicles have been explored as effective materials for preventing colorectal cancer (CRC) incidence, dependent on gene status, as a K-Ras-activating mutation via the macropinocytosis pathway. Approximately 70% of CRC harbors the p53 mutation, which is strongly associated with a poor prognosis for CRC. However, it has not been revealed whether p53 inactivation activates the macropinocytosis pathway or not. In this study, we investigated parental cells, wild-type or null for p53 treated with Citrus limon L.-derived nanovesicles, as potential materials for CRC prevention. Using ultracentrifugation, we obtained C. limon L.-derived nanovesicles, the diameters of which were approximately 100 nm, similar to that of the exosomes derived from mammalian cells. C. limon L.-derived nanovesicles showed inhibitory effects on cell growth in not p53-wild, but also in p53-inactivated CRC cells. Furthermore, we revealed that the macropinocytosis pathway is activated by p53 inactivation and C. limon L.-derived nanovesicles were up taken via the macropinocytosis pathway. Notably, although C. limon L.-derived nanovesicles contained citrate, the inhibitory effects of citrate were not dependent on the p53 status. We thus provide a novel mechanism for the growth inhibition of C. limon L.-derived nanovesicles via macropinocytosis and expect to develop a functional food product containing them for preventing p53-inactivation CRC incidence.

Sodium salicylate and 5-aminosalicylic acid synergistically inhibit the growth of human colon cancer cells and mouse intestinal polyp-derived cells.

As colon cancer is one of the most common cancers in the world, practical prevention strategies for colon cancer are needed. Recently, treatment with aspirin and/or 5-aminosalicylic acid-related agents was reported to reduce the number of intestinal polyps in patients with familial adenomatous polyposis. To evaluate the mechanism of aspirin and 5-aminosalicylic acid for suppressing the colon polyp growth, single and combined effects of 5-aminosalicylic acid and sodium salicylate (metabolite of aspirin) were tested in the two human colon cancer cells with different cyclooxygenase-2 expression levels and intestinal polyp-derived cells from familial adenomatous polyposis model mouse. The combination induced cell-cycle arrest at the G1 phase along with inhibition of cell growth and colony-forming ability in these cells. The combination reduced cyclin D1 via proteasomal degradation and activated retinoblastoma protein. The combination inhibited the colony-forming ability of mouse colonic mucosa cells by about 50% and the colony-forming ability of mouse intestinal polyp-derived cells by about 90%. The expression level of cyclin D1 in colon mucosa cells was lower than that in intestinal polyp-derived cells. These results suggest that this combination may be more effective in inhibiting cell growth of intestinal polyps through cyclin D1 down-regulation.

A chemoproteoinformatics approach demonstrates that aspirin increases sensitivity to MEK inhibition by directly binding to RPS5.

MEK inhibitors are among the most successful molecularly targeted agents used as cancer therapeutics. However, to treat cancer more efficiently, resistance to MEK inhibitor-induced cell death must be overcome. Although previous genetic approaches based on comprehensive gene expression analysis or RNAi libraries led to the discovery of factors involved in intrinsic resistance to MEK inhibitors, a feasible combined treatment with the MEK inhibitor has not yet been developed. Here, we show that a chemoproteoinformatics approach identifies ligands overcoming the resistance to cell death induced by MEK inhibition as well as the target molecule conferring this resistance. First, we used natural products, perillyl alcohol and sesaminol, which induced cell death in combination with the MEK inhibitor trametinib, as chemical probes, and identified ribosomal protein S5 (RPS5) as their common target protein. Consistently, trametinib induced cell death in RPS5-depleted cancer cells via upregulation of the apoptotic proteins BIM and PUMA. Using molecular docking and molecular dynamics (MD) simulations, we then screened FDA- and EMA-approved drugs for RPS5-binding ligands and found that acetylsalicylic acid (ASA, also known as aspirin) directly bound to RPS5, resulting in upregulation of BIM and PUMA and induction of cell death in combination with trametinib. Our chemoproteoinformatics approach demonstrates that RPS5 confers resistance to MEK inhibitor-induced cell death, and that aspirin could be repurposed to sensitize cells to MEK inhibition by binding to RPS5.

Oridonin inhibits SASP by blocking p38 and NF-κB pathways in senescent cells.

Cellular senescence is a state of irreversible cell growth arrest that functions as a biological defense mechanism against severe DNA damage. Senescent cells with DNA damage produce pro-inflammatory cytokines, such as IL-6 and IL-8, and this phenomenon is called the senescence-associated secretory phenotype (SASP). SASP factors have been implicated in various disorders, including cancer. We performed a screening assay and identified oridonin as a candidate SASP inhibitor. Oridonin is an active diterpenoid that is isolated from Isodon plants and has been reported to exhibit anti-inflammatory, antibacterial, antioxidant, and antitumor activities. It reduced the secretion of IL-6 and IL-8 in senescent cells at the protein and mRNA levels. Oridonin also inhibited p65 subunit of NF-κB activity. However, oridonin did not affect SA ß-gal activity and enhanced the expression of p21. The expression and phosphorylation of p38 were down-regulated by oridonin. The p38 inhibitor SB203580 inhibited the secretion of IL-8, slightly inhibited the secretion of IL-6, and did not affect NF-κB activity. Therefore, the NF-κB and p38 pathways may contribute to the inhibition of SASP by oridonin. Oridonin has potential as a therapeutic agent for SASP-related diseases.

Rabdosianone I, a Bitter Diterpene from an Oriental Herb, Suppresses Thymidylate Synthase Expression by Directly Binding to ANT2 and PHB2.

Natural products have numerous bioactivities and are expected to be a resource for potent drugs. However, their direct targets in cells often remain unclear. We found that rabdosianone I, which is a bitter diterpene from an oriental herb for longevity, Isodon japonicus Hara, markedly inhibited the growth of human colorectal cancer cells by downregulating the expression of thymidylate synthase (TS). Next, using rabdosianone I-immobilized nano-magnetic beads, we identified two mitochondrial inner membrane proteins, adenine nucleotide translocase 2 (ANT2) and prohibitin 2 (PHB2), as direct targets of rabdosianone I. Consistent with the action of rabdosianone I, the depletion of ANT2 or PHB2 reduced TS expression in a different manner. The knockdown of ANT2 or PHB2 promoted proteasomal degradation of TS protein, whereas that of not ANT2 but PHB2 reduced TS mRNA levels. Thus, our study reveals the ANT2- and PHB2-mediated pleiotropic regulation of TS expression and demonstrates the possibility of rabdosianone I as a lead compound of TS suppressor.

Artesunate inhibits intestinal tumorigenesis through inhibiting wnt signaling.

Artesunate (ART) is a clinically approved antimalarial drug and was revealed as a candidate of colorectal cancer chemopreventive agents in our drug screening system. Here, we aimed to understand the suppressive effects of ART on intestinal tumorigenesis. In vitro, ART reduced T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter transcriptional activity. In vivo, ART inhibited intestinal polyp development. We found that ART reduces TCF1/TCF7 nuclear translocation by binding the Ras-related nuclear protein (RAN), suggesting that ART inhibits TCF/LEF transcriptional factor nuclear translocation by binding to RAN, thereby inhibiting Wnt signaling. Our results provide a novel mechanism through which artesunate inhibits intestinal tumorigenesis.

Synergistic effect of the inhibitors of RAF/MEK and AXL on KRAS-mutated ovarian cancer cells with high AXL expression.

KRAS mutation is frequently seen in a subtype of ovarian cancer categorized as type 1. The KRAS-MAPK pathway, which is closely involved in type 1 cancer progression, is under the regulation of receptor tyrosine kinases (RTKs). AXL, one of the RTKs, has been reported to be overexpressed in ovarian cancer and contributes to the poor prognosis. However, there is no useful target-based agent against such gene profiles. We examined the combined effect of the dual RAF/MEK inhibitor CH5126766 and AXL inhibitor R428 on the growth of ovarian cancer HEY-T30 and OVCAR-5 cell lines, both of which bear KRAS mutation and express AXL at a high level, using the WST-8 assay and the colony formation assay. The synergistic effect of the combination was evaluated by the combination index. The apoptotic cells were analyzed by flow cytometry. The expression of apoptotic proteins and the phosphorylation of MAPK and AKT pathway proteins were investigated by western blotting. We found that CH5126766 and R428 suppressed the phosphorylation of ERK and AKT, respectively, and their combination synergistically inhibited the growth of both cell lines with enhancement of apoptosis accompanied by the Bim upregulation. Combined treatment with CH5126766 and R428 is expected as the novel therapeutic option for KRAS-mutated ovarian cancer with high expression of AXL.

Histone deacetylase inhibitor OBP­801 and amrubicin synergistically inhibit the growth of squamous cell lung carcinoma by inducing mitochondrial ASK1­dependent apoptosis.

Squamous cell lung carcinoma (SQCLC) is an aggressive type of lung cancer. In contrast with the marked advances that have been achieved in the treatment of lung adenocarcinoma, there are currently no effective targeted therapies for SQCLC, for with cytotoxic drugs are still the main treatment strategy. Therefore, the present study aimed to develop novel combination therapies for SQCLC. The results demonstrated that a combined treatment with the potent histone deacetylase (HDAC) inhibitor OBP­801 and the third­generation anthracycline amrubicin synergistically inhibited the viability of SQCLC cell lines by inducing apoptosis signal­regulating kinase 1 (ASK1)­dependent, as well as JNK­ and p38 mitogen­activated protein kinase (MAPK)­independent apoptosis. OBP­801 treatment strongly induced the protein expression levels of thioredoxin­interacting protein (TXNIP), and amrubicin treatment increased the levels of intracellular reactive oxygen species (ROS), which suggested that this combination oxidized and dissociated thioredoxin 2 (Trx2) from mitochondrial ASK1 and activated ASK1. Moreover, mouse xenograft experiments using human H520 SQCLC cells revealed that the co­treatment potently suppressed tumor growth in vivo. These results suggested that a combined treatment with OBP­801 and amrubicin may have potential as a therapeutic strategy for SQCLC.

Sulindac sulfone inhibits the mTORC1 pathway in colon cancer cells by directly targeting voltage-dependent anion channel 1 and 2.

Sulindac sulfone is a metabolite of sulindac, a non-steroidal anti-inflammatory drug (NSAID), without anti-inflammatory ability. However, sulindac sulfone has been reported to significantly reduce polyps in patients with colorectal adenomatous polyposis in clinical trials. Thus, sulindac sulfone is expected to be useful for the chemoprevention of neoplasia with few side effects related to anti-inflammatory ability. To date, the molecular targets of sulindac sulfone have not yet fully investigated. Therefore, in order to newly identify sulindac sulfone-binding proteins, we generated sulindac sulfone-fixed FG beads and purified sulindac sulfone-binding proteins from human colon cancer HT-29 cells. we identified mitochondrial outer membrane proteins voltage-dependent anion channel (VDAC) 1 and VDAC2 as novel molecular targets of sulindac sulfone, and sulindac sulfone directly bound to both VDAC1 and VDAC2. Double knockdown of VDAC1 and VDAC2 by siRNA inhibited growth and arrested the cell cycle at G1 phase in HT-29 cells. Depletion of VDAC1 and VDAC2 also inhibited the mTORC1 pathway with a reduction in cyclin D1. Interestingly, these effects were consistent with those of sulindac sulfone against human colon cancer cells, suggesting that sulindac sulfone negatively regulates the function of VDAC1 and VDAC2. In the present study, our data suggested that VDAC1 and VDAC2 are direct targets of sulindac sulfone which suppresses the mTORC1 pathway and induces G1 arrest.

The histone deacetylase inhibitor OBP-801 and eribulin synergistically inhibit the growth of triple-negative breast cancer cells with the suppression of survivin, Bcl-xL, and the MAPK pathway.

PURPOSE: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Eribulin was approved for the treatment of metastatic breast cancer through the EMBRACE trial, and a subgroup analysis in this clinical trial indicated the efficacy of eribulin in patients with TNBC. However, the prognosis of patients with TNBC is still poor due to various molecular characteristics. Therefore, there is an urgent need for a more effective treatment for the management of TNBC. METHODS: We investigated the synergistic effect of a novel histone deacetylase (HDAC) inhibitor, OBP-801, and eribulin in TNBC cell lines because OBP-801 has been known to enhance the anti-tumor activities of other chemotherapeutic agents. The cell growth was analyzed, and the flow cytometry analysis was conducted to evaluate the effects on cell cycle and the induction of apoptosis. The mechanism underlying the enhancement of inhibition of TNBC cell growth was investigated through Western blot analyses. RESULTS: The combination treatment of OBP-801 with eribulin showed the synergistic inhibition of the growth in TNBC cells, involved with the enhancement of apoptosis. We, for the first time, found that eribulin upregulated survivin and also that OBP-801 could remarkably suppress the upregulation of survivin by eribulin. Moreover, this combination potently suppressed Bcl-xL and the MAPK pathway compared with either agent alone. CONCLUSION: We found that the combination of OBP-801 and eribulin synergistically inhibited the growth with apoptosis in TNBC cells, suggesting that this combination might be a promising novel strategy for treating TNBC patients.

Ribosomal protein S3 regulates XIAP expression independently of the NF-κB pathway in breast cancer cells.

The X-linked inhibitor of apoptosis (XIAP) confers the resistance of various types of cancer to standard chemotherapeutic agents such as anthracycline and taxane. In breast cancer, XIAP is known to be overexpressed. However, the mechanisms underlying the overexpression of XIAP remain currently unclear. In order to elucidate the mechanisms responsible for the overexpression of the XIAP protein in breast cancer, we attempted to clarify the mechanisms by which the natural compound curcumin downregulates XIAP in breast cancer cells. In that process, we identified the ribosomal protein S3 (RPS3) as a curcumin­binding protein using curcumin-fixed magnetic FG beads. The knockdown of RPS3 inhibited cell growth and induced apoptosis as well as the downregulation of XIAP in breast cancer cells. Although RPS3 is known to directly bind to and activate the nuclear factor-κB (NF-κB), which induces several anti-apoptotic genes such as XIAP, the knockdown of RPS3 unexpectedly reduced the levels of the XIAP protein, but not the mRNA level of XIAP and the transcription factor NF-κB activity. These results reveal that RPS3 upregulates XIAP independently of the NF-κB pathway in human breast cancer cells.

Resibufogenin Induces G1-Phase Arrest through the Proteasomal Degradation of Cyclin D1 in Human Malignant Tumor Cells.

Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB) protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3ß (GSK-3ß) induces the proteasomal degradation of cyclin D1. The addition of GSK-3ß inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3ß.

The alkaloid emetine sensitizes ovarian carcinoma cells to cisplatin through downregulation of bcl-xL.

Cisplatin and its platinum derivatives are first-line chemotherapeutic agents in the treatment of ovarian cancer. However, chemoresistance is the leading cause of therapeutic failure and is responsible for the poor overall survival rate. Here, we describe that emetine, a natural alkaloid used as an anti-amoebiasis drug, sensitized ovarian carcinoma cells to apoptosis induced by cisplatin. The single administration of cisplatin or emetine had a weak effect on cell death. However, co-treatment of cisplatin and emetine remarkably induced apoptosis and reduced the colony formation of ovarian carcinoma cells. Moreover, we showed that apoptosis induced by the combination of cisplatin and emetine was dependent on the activation of caspases -3, -7 and -8. As to the mechanism, downregulation of bcl-xL by emetine was shown to be responsible for enhancing the sensitivity of ovarian cancer cells to cisplatin. These findings suggest that the combination of cisplatin and emetine might be a promising treatment for ovarian cancer.

The flavonoid apigenin downregulates CDK1 by directly targeting ribosomal protein S9.

Flavonoids have been reported to inhibit tumor growth by causing cell cycle arrest. However, little is known about the direct targets of flavonoids in tumor growth inhibition. In the present study, we developed a novel method using magnetic FG beads to purify flavonoid-binding proteins, and identified ribosomal protein S9 (RPS9) as a binding partner of the flavonoid apigenin. Similar to treatment with apigenin, knockdown of RPS9 inhibited the growth of human colon cancer cells at the G2/M phase by downregulating cyclin-dependent kinase 1 (CDK1) expression at the promoter level. Furthermore, knockdown of RPS9 suppressed G2/M arrest caused by apigenin. These results suggest that apigenin induces G2/M arrest at least partially by directly binding and inhibiting RPS9 which enhances CDK1 expression. We therefore raise the possibility that identification of the direct targets of flavonoids may contribute to the discovery of novel molecular mechanisms governing tumor growth.

Vesnarinone suppresses TNFα mRNA expression by inhibiting valosin-containing protein.

Vesnarinone is a synthetic quinolinone derivative used in the treatment of cardiac failure and cancer. It is also known to cause agranulocytosis as a side effect, which restricts its use, although the mechanism underlying agranulocytosis is not well understood. Here, we show that vesnarinone binds to valosin-containing protein (VCP), which interacts with polyubiquitinated proteins and is essential for the degradation of IκBα to activate nuclear factor (NF)κB. We show that vesnarinone impairs the degradation of IκBα, and that the impairment of the degradation of IκBα is the result of the inhibition of the interaction between VCP and the 26S proteasome by vesnarinone. These results suggest that vesnarinone suppresses NFκB activation by inhibiting the VCP-dependent degradation of polyubiquitinated IκBα, resulting in the suppression of tumor necrosis factor-α mRNA expression.

Apigenin sensitizes prostate cancer cells to Apo2L/TRAIL by targeting adenine nucleotide translocase-2.

Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. Recombinant human Apo2L/TRAIL has been under clinical trials, whereas various kinds of malignant tumors have resistance to Apo2L/TRAIL. We and others have shown that several anticancer agents and flavonoids overcome resistance to Apo2L/TRAIL by upregulating death receptor 5 (DR5) in malignant tumor cells. However, the mechanisms by which these compounds induce DR5 expression remain unknown. Here we show that the dietary flavonoid apigenin binds and inhibits adenine nucleotide translocase-2 (ANT2), resulting in enhancement of Apo2L/TRAIL-induced apoptosis by upregulation of DR5. Apigenin and genistein, which are major flavonoids, enhanced Apo2L/TRAIL-induced apoptosis in cancer cells. Apigenin induced DR5 expression, but genistein did not. Using our method identifying the direct targets of flavonoids, we compared the binding proteins of apigenin with those of genistein. We discovered that ANT2 was a target of apigenin, but not genistein. Similarly to apigenin, knockdown of ANT2 enhanced Apo2L/TRAIL-induced apoptosis by upregulating DR5 expression at the post-transcriptional level. Moreover, silencing of ANT2 attenuated the enhancement of Apo2L/TRAIL-induced apoptosis by apigenin. These results suggest that apigenin upregulates DR5 and enhances Apo2L/TRAIL-induced apoptosis by binding and inhibiting ANT2. We propose that ANT2 inhibitors may contribute to Apo2L/TRAIL therapy.

Perillyl alcohol causes G1 arrest through p15(INK4b) and p21(WAF1/Cip1) induction.

The monoterpene perillyl alcohol (POH) is a naturally occurring compound derived from citrus fruits, mint and herbs. It exhibited chemotherapeutic potential against various malignant tumors in preclinical models and is currently being tested in clinical trials in patients with refractory advanced cancers. POH inhibits cellular proliferation at the G1 phase of the cell cycle in vitro. However, the molecular mechanisms responsible for this effect have not been sufficiently elucidated. Here we showed that 1.0 mM POH upregulates p15(INK4b) and p21(WAF1/Cip1), resulting in hypophosphorylation of the retinoblastoma (RB) protein and subsequent G1 arrest in human immortalized keratinocyte HaCaT cells. The induction of p15(INK4b) was mediated through its promoter, but that of p21(WAF1/Cip1) was not. The small interfering RNA (siRNA) of either p15(INK4b) or p21(WAF1/Cip1) significantly attenuated the increase in the G1 cell population caused by POH. The induction of p15(INK4b) and p21(WAF1/Cip1) and sub-sequent G1 arrest by POH was also observed in other cancer cell lines. These results suggest that the induction of p15(INK4b) as well as p21(WAF1/Cip1) is associated with the antiproliferative effect of POH.