Solanoeclepin B, a hatching factor for potato cyst nematode.

The potato cyst nematode (PCN) causes extensive crop losses worldwide. Because the hatching of PCN requires host-derived molecules known as hatching factors (HFs), regulating HF production in host plants may help to control this harmful pest. Solanoeclepin A (SEA), isolated from potato, is the most active HF for PCN; however, its biosynthesis is completely unknown. We discovered a HF called solanoeclepin B (SEB) from potato and tomato root exudates and showed that SEB was biosynthesized in the plant and converted to SEA outside the plant by biotic agents. Moreover, we identified five SEB biosynthetic genes encoding three 2-oxoglutarate-dependent dioxygenases and two cytochrome P450 monooxygenases in tomato. Exudates from tomato hairy roots in which each of the genes was disrupted contained no SEB and had low hatch-stimulating activity for PCN. These findings will help to breed crops with a lower risk of PCN infection.

A case of microsatellite instability-high clinically advanced castration-resistant prostate cancer showing a remarkable response to pembrolizumab sustained over at least 18 months.

Defective DNA mismatch repair genes can lead to microsatellite instability (MSI)-high status in prostate cancer (PC). Accumulation of replication errors in DNA leads to the production of abundant neoantigens, which could be targets for immune checkpoint inhibitors (CPIs). However, the incidence of MSI-high PC is low, and not all patients show a satisfactory therapeutic response to CPIs. Here, we present the case of a patient with MSI-high castration-resistant PC who showed a remarkable and durable response to pembrolizumab. The patient was resistant to abiraterone, docetaxel, and cabazitaxel and was suffering from multiple tumor-associated or treatment-related complications, such as urinary tract infection, infective endocarditis, and uncontrollable prostatic hemorrhage. Soon after the start of pembrolizumab therapy, the patient showed a dramatic decrease in prostate-specific antigen from 35.67 ng/mL to an undetectable level and a remarkable reduction in the size of a massive prostate mass and lymph node metastases, with an absence of treatment-related complications. Specimens from the transurethral resection of prostate cancer during cabazitaxel treatment for control of prostate bleeding and also that from the prostate biopsy at initial diagnosis revealed MSI-high status. Immunohistochemistry showed loss of MSH2 and MSH6, and whole-exome sequencing revealed an approximate tumor mutation burden of 61 mutations/Mb as well as biallelic loss of MSH2 Pembrolizumab could show a significant effect even in a heavily treated patient with MSI-high advanced PC. Accumulation of detailed clinical and genomic information of cases of MSI-high PC treated with pembrolizumab is necessary for optimal patient selection.

Use of positron emission tomography for real-time imaging of biodistribution of green tea catechin.

The aim of this study was to achieve real-time imaging of the in vivo behavior of a green tea polyphenol, catechin, by positron emission tomography (PET). Positron-labeled 4″ -[(11)C]methyl-epigallocatechin gallate ([(11)C]Me-EGCG) was orally administered to rats, and its biodistribution was imaged for 60 min by using a small animal PET system. As the result, images of [(11)C]Me-EGCG passing through the stomach into the small intestines were observed; and a portion of it was quantitatively detected in the liver. On the other hand, intravenous injection of [(11)C]Me-EGCG resulted in a temporal accumulation of the labeled catechin in the liver, after which almost all of it was transferred to the small intestines within 60 min. In the present study, we succeeded in obtaining real-time imaging of the absorption and biodistribution of [(11)C]Me-EGCG with a PET system.

Inhibitory effect of liposomal rhinacanthin-N isolated from Rhinacanthus nasutus on pulmonary metastasis in mice.

We previously observed that rhinacanthins, which are the main naphthoquinone esters isolated from the roots of a Thai medicinal plant, Rhinacanthus nasutus KURZ. (family Acanthaceae), suppress the growth of Meth-A sarcoma in the tumor-bearing mice and that rhinacanthin-N has the strongest antitumor activity among these naphthoquinone esters tested. In the present study, we investigated the effect of rhinacanthin-N on pulmonary metastasis induced by B16F10 melanoma cells in mice. C57BL/6 male mice were injected intravenously with B16F10 melanoma cells, and liposomal rhinacanthin-N was administered intraperitoneally from day 1 to 7 after tumor implantation. Liposomes were used to formulate an injectable form of the hydrophobic agent. Treatment of the mice with 5 or 10 mg/kg/d of liposomal rhinacanthin-N significantly inhibited the pulmonary metastatic colonization of the melanoma cells. Based on these data, our findings demonstrate that rhinacanthin-N possesses antimetastatic efficacy, which may make it a lead compound for the development of a new anticancer drug for use in cancer chemotherapy.

Anti-angiogenic activity and intracellular distribution of epigallocatechin-3-gallate analogs.

Angiogenesis, a process of construction of new blood capillaries, is crucial for tumor progression and metastasis. Our previous studies demonstrated that a component of green tea, epigallocatechin-3-gallate (EGCG), suppressed angiogenesis and subsequent tumor growth. In this study, to elucidate the detailed mechanism of the anti-angiogenic effect of EGCG and to enhance the antiangiogenic activity of EGCG, we designed and synthesized EGCG derivatives and examined their biological effect and intracellular localization in human umbilical vein endothelial cells (HUVECs). EGCG derivatives aminopentyl dideoxyEGCG and aminopentyl dideoxygallocatechin-3-gallate (cis-APDOEGCG and trans-APDOEGCG) had an enhanced inhibitory effect on the proliferation when used at more than 30 µM. To elucidate antiangiogenic effect of EGCG, we used a 1 µM concentration for subsequent experiments where no effect on proliferation was observed. These EGCG derivatives showed a stronger inhibitory effect on migration, invasion, and tube formation by HUVECs than the non-derivatized EGCG. Furthermore, the derivatives induced a change in the distribution of F-actin and subsequent morphology of the HUVECs. Next, we synthesized fluorescent TokyoGreen-conjugated EGCG derivative (EGCG-TG) and observed the distribution in HUVECs under a confocal laser scanning microscope. Abundant fluorescence was observed in the cells after a 3-h incubation, and was localized in mitochondria as well as in cytoplasm. These results suggest that EGCG was incorporated into the HUVECs, that a portion of it entered into their mitochondria.

Concise synthesis of catechin probes enabling analysis and imaging of EGCg.

A concise synthesis of APDOEGCg (3) was accomplished. Due to the reactivity of its amine group, the compound could be easily converted to the fluorescein probe 21 and immunogen probe 22 efficiently. We then demonstrated the usefulness of the probes for imaging studies and the generation of antibodies.

Preventive effect of green tea catechins on experimental tumor metastasis in senescence-accelerated mice.

Successful avoidance of the immune surveillance system is critical for the development of a blood-borne metastasis. Previous findings suggest that experimental tumor metastasis was enhanced in senescence-accelerated mice prone 10 (SAMP10) due to a reduction in immune surveillance potential with age. In the present study, water containing green tea (GT)-catechins was freely given to SAMP10 mice, and the chemopreventive effect of GT-catechin intake on tumor metastasis was examined. Natural killer cell activity, which is an indicator of immune surveillance potential and is reduced in control mice with age, was maintained by GT-catechin intake. The early accumulation of lung-metastatic K1735M2 melanoma cells in lungs after intravenous injection of the cells and subsequent experimental lung metastasis was investigated in mice given GT-catechins. The accumulation at 6 and 24 h after injection of K1735M2 cells was significantly suppressed, and the number of lung-metastatic colonies was significantly reduced, in comparison with those in control mice. The results suggest that GT-catechin intake prevented the experimental tumor metastasis in aged SAMP10 mice via its inhibition of a reduction in immune surveillance potential with age.

Antitumor activity of liposomal naphthoquinone esters isolated from Thai medicinal plant: Rhinacanthus nasutus KURZ.

We previously observed that rhinacanthins-C, -N and -Q, three main naphthoquinone esters isolated from the roots of Thai medicinal plant; Rhinacanthus nasutus KURZ. (Acanthaceae) induced apoptosis of human cervical carcinoma HeLaS3 cells. Since these rhinacanthins showed limited solubility in aqueous medium, we attempted to entrap them into liposomal membrane: Liposomalization enabled injection of the drugs and the drugs were expected to transfer to lipoproteins in the bloodstream. Liposomal formulations of rhinacanthins-C, -N and -Q showed strong antiproliferative activity against HeLaS3 cells with the IC50 values of 32, 17, 70 microM; 19, 17, 52 microM and 2.7, 2.0 and 5.0 microM for the exposure time of 24, 48, and 72 h, respectively. These liposomes suppressed the tumor growth in Meth-A sarcoma-bearing BALB/c mice at the dose of 5.0 mg/kg/d for 10 d. Among rhinacanthins, liposomal rhinacanthin-N significantly suppressed solid tumor growth. Based on these results, our findings demonstrated that rhinacanthin-N suppressed tumor growth in vivo, and suggested that liposomes are useful for preparing injectable formulation of hydrophobic drugs.

Induction of apoptosis in tumor cells by three naphthoquinone esters isolated from Thai medicinal plant: Rhinacanthus nasutus KURZ.

Rhinacanthus nasutus KURZ. (Acanthaceae) has been used as Thai traditional medicine for the treatment of various cancers. Recently, we reported that rhinacanthins, active components of the plant, had antiproliferative activity against human cancer line cells. In the present study, we investigated the growth inhibitory mechanism of rhinacanthins-C, -N and -Q, three main naphthoquinone esters isolated from the roots of R. nasutus KURZ. in human cervical carcinoma (HeLaS3) cells by means of TUNEL staining, DNA fragmentation assay, flow cytometry, and cleavage assay of Asp-Glu-Val-Asp-peptide-nitroanilide, a caspase-3 substrate. After the HeLaS3 cells was exposed with different concentrations of the drugs, rhinacanthins-C, -N and -Q exhibited antiproliferative effects on HeLaS3 cells with the IC50 values of 80, 65, 73 microM; 55, 45, 55 microM; and 1.5, 1.5 and 5.0 microM for 24, 48 and 72 h time points, respectively. Morphological changes showing nuclear fragmentation of rhinacanthins-treated cells were clearly observed after 48 h exposure. Consistent with this observation, the appearance of a ladder formation was also evident with an agarose gel electrophoresis of the extracted DNA. Flow cytometric analysis revealed that rhinacanthin-N caused G2/M arrest of HeLaS3 cells after 24 h incubation, and increased the proportion of sub-G1 hypodiploid cells, apoptotic cells, in the population of HeLaS3 cells after 48 and 72 h incubation. Moreover, the drug treatment markedly elevated the activity of caspase-3. Based on these results, our findings demonstrated for the first time that the inhibitory effects of three main naphthoquinone esters isolated from the roots of R. nasutus KURZ. on the growth of HeLaS3 cells appear to arise from the induction of apoptosis, that might be associated with the activation of caspase-3 pathway.