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.

Chemotherapy for colorectal cancer has become more complicated and diversified with the appearance of molecular‐targeting agents. 5‐Fluorouracil (5‐FU) has been a mainstay of chemotherapy for colorectal cancer, but it is still unknown whether the combining of 5‐FU with novel molecular‐targeting agents is effective. Thymidylate synthase (TS) is a direct target of 5‐FU, and the low TS level has been generally supposed to sensitize 5‐FU's efficacy. We therefore hypothesized that RB‐reactivating agents could enhance the efficacy of 5‐FU, because the RB‐reactivating agents could suppress the function of transcription factor E2F of TS gene promoter. We used three RB‐reactivating agents, trametinib/GSK1120212 (MEK inhibitor), fenofibrate (PPARα agonist), and LY294002 (PI3K inhibitor), with 5‐FU against human colon cancer HT‐29 and HCT15 cells. Trametinib induced p15 and p27 expression and reduced cyclin D1 levels in HT‐29 cells. Fenofibrate also dephosphorlated ERK1/2 and reduced cyclin D1 levels in HT‐29 cells. LY294002 induced p27 expression in HCT15 cells. All three agents caused dephosphorylation of RB protein and G1‐phase arrest with a reduction of TS expression. As a consequence, the combination of 5‐FU with each of the agents resulted in a significant decrease of colony numbers in HT‐29 or HCT15 cells. These results suggest “RB‐reactivation therapy” using molecular‐targeting agents to be a new strategy for 5‐FU‐based chemotherapy. In particular, we strongly expect trametinib, which was discovered in Japan and was recently submitted to FDA for approval, to be used together with established regimens for colorectal cancer.

As histone deacetylase inhibitors (HDACIs) have limited efficacy against solid tumors, we investigated whether and how oxidative stress is involved in sensitivity to HDACIs to develop a novel therapeutic option of HDACIs treatment. We first tested whether a reduction of the antioxidant glutathione (GSH) by glutamine deprivation affects sensitivity to a commercially available HDACI vorinostat and reactive oxygen species (ROS) accumulation. Next we investigated the relationship between a glutamate-cystine transporter xCT and the efficacy of vorinostat using siRNA of xCT and bioinformatic analyses. Finally, we verified the combinatory effects of vorinostat and the xCT inhibitor salazosulfapyridine (SASP) on ROS accumulation, cell death induction, and colony formation. Glutamine deprivation increased vorinostat-mediated cell death with ROS accumulation. Genetic ablation of xCT improved the efficacy of vorinostat, consistent with the results of public data analyses demonstrating that xCT expressions positively correlate with insensitivity to HDACIs in many types of cancer cell lines. Vorinostat caused ROS accumulation when combined with SASP, possibly resulting in synergistic ferroptosis. Our study provides a novel mechanistic insight into the mechanism underlying sensitivity to HDACIs involving xCT, suggesting xCT to be a promising predictive marker of HDACIs and rationalizing combinatory therapy of HDACIs with xCT inhibitors to induce ferroptosis.

The expression of cyclin D1 is upregulated in various cancer cells by diverse mechanisms, such as increases in mRNA levels, the promotion of the translation by mammalian target of rapamycin complex 1 (mTORC1) signaling and the protein stabilization. We here show that sesaminol, a sesame lignan, reduces the expression of cyclin D1 with decreasing mRNA expression levels, inhibiting mTORC1 signaling and promoting proteasomal degradation. We subsequently generated sesaminol-immobilized FG beads to newly identify sesaminol-binding proteins. As a consequence, we found that adenine nucleotide translocase 2 (ANT2), the inner mitochondrial membrane protein, directly bound to sesaminol. Consistent with the effects of sesaminol, the depletion of ANT2 caused a reduction in cyclin D1 with decreases in its mRNA levels, mTORC1 inhibition and the proteasomal degradation of its protein, suggesting that sesaminol negatively regulates the function of ANT2. Furthermore, we screened other ANT2-binding compounds and found that the proliferator-activated receptor-γ agonist troglitazone also reduced cyclin D1 expression in a multifaceted manner, analogous to that of the sesaminol treatment and ANT2 depletion. Therefore, the chemical biology approach using magnetic FG beads employed in the present study revealed that sesaminol bound to ANT2, which may pleiotropically upregulate cyclin D1 expression at the mRNA level and protein level with mTORC1 activation and protein stabilization. These results suggest the potential of ANT2 as a target against cyclin D1-overexpressing cancers.

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.