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.

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-3β (GSK-3β) induces the proteasomal degradation of cyclin D1. The addition of GSK-3β 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-3β.

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.

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.

In a standard free jejunal transfer, one artery and one vein are anastomosed. However, when raising the jejunal flap, a one-segment jejunum sometimes has two arteries and one accompanying vein as a vascular pedicle. Free jejunal transfer in which two arteries and one vein are anastomosed was designed. We report on the safety and advantages of using this artery-dominant transfer when performing microvascular anastomosis. This technique was used when a one-segment jejunum had two arteries and an accompanying vein. Eight patients underwent this arterial-supercharged free jejunal transfer. All flaps survived, and no complications developed except for two cases of intraoperative thrombosis before the procedure. It is important to transfer the artery-rich graft into the same physiological environment by reconstructing the similar hemodynamics. The grafts can be transferred without harm. This artery-dominant method can be an option when conditions are unfavorable for safer jejunal transfer.