Retro-aza-Michael reaction of an o-aminophenol adduct in protic solvents inspired by natural products.

α,ß-Unsaturated carbonyls are reactive group often found in bioactive small molecules. Their non-specific reaction with biomolecules can be the cause of the low efficacy and unexpected side-effects of the molecule. Accordingly, unprotected α,ß-unsaturated carbonyls are not often found in drugs. Here, we report that o-aminophenol is a new masking group of α,ß-unsaturated ketone, which is inspired by natural products saccharothriolides. o-Aminophenol adduct of α,ß-unsaturated ketone, but not those of α,ß-unsaturated amide or ester, undergoes a retro-Michael reaction to yield o-aminophenol and the Michael acceptor. This reaction was observed only in protic solvents, such as MeOH and aqueous MeOH. In contrast, o-anisidine was not eliminated from its Michael adduct. o-Aminophenol may be a promising masking tool of highly-reactive bioactive α,ß-unsaturated carbonyl compounds.

Two New Halogenated Compounds from the Marine Red Alga Laurencia nipponica Yamada from the Kunashiri and Etorofu Islands.

The red alga Laurencia nipponica comprises various chemical races distributed relative to the ocean current in Japanese coastal areas. We investigated the chemical compositions and chemical races of L. nipponica distributed from the Kunashiri and Etorofu Islands, the confluence of the Soya warm current and Oya-shio cold current. Two new halogenated secondary metabolites, deacetylneonipponallene (1) and neopacifenol (2), along with four known compounds, deoxyprepacifenol (3), pacifenol (4), halo-chamigrene diether (5), and isolaurallene (6) were isolated from L. nipponica collected at Chikappunai, Kunashiri Island, while Zaimokuiwa (Kunashiri Island) and Sana (Etorofu Island) populations contained 3, 7-hydroxylaurene (7), 2,10-dibromo-3-chloro-9-hydroxy-α-chamigrene (8), and (3Z)-laurefucin (9). The structures of 1 and 2 were established using spectroscopic methods. The chemical races of L. nipponica distributed in this area were divided into 6- and 9-producing races. Interestingly, both races contained 4 as an additional race-index, as well as its derivatives, 2 and 5. To the best of our knowledge, this is the first example of a race comprising a mixture of two race-index compounds, suggesting that the convergence of two currents causes the production of new and diverse chemical races in this species.

Total Synthesis and Antimicrobial Activity of Tumescenamide C and Its Derivatives.

Tumescenamide C (1) is an antimicrobial compound produced by Streptomyces sp. KUSC_F05 and consists of a cyclic depsipeptide core and a polyketide side chain with branched methyl groups. Here, we report the total synthesis of tumescenamide C and two derivatives, mainly using Fmoc solid-phase peptide synthesis (SPPS). In addition, a biological evaluation of these compounds revealed the critical partial structure in 1 for antimicrobial activity.

New Marine Antifouling Compounds from the Red Alga Laurencia sp.

Six new compounds, omaezol, intricatriol, hachijojimallenes A and B, debromoaplysinal, and 11,12-dihydro-3-hydroxyretinol have been isolated from four collections of Laurencia sp. These structures were determined by MS and NMR analyses. Their antifouling activities were evaluated together with eight previously known compounds isolated from the same samples. In particular, omaezol and hachijojimallene A showed potent activities (EC50 = 0.15-0.23 µg/mL) against larvae of the barnacle Amphibalanus amphitrite.

Clinical significance of mucinous components in rectal cancer after preoperative chemoradiotherapy.

PURPOSE: The clinical implications of mucinous components in rectal tumors, especially with regard to the efficacy of neoadjuvant chemoradiotherapy, remain unclear. METHODS: One hundred and thirty rectal cancer patients who received curative resection after neoadjuvant chemoradiotherapy were retrospectively reviewed. Patients were classified into 3 groups according to the proportion of extracellular mucin: low (<5 %), moderate (5-25 %), and high (>25 %). RESULTS: There were 82 (63.1 %), 26 (20.0 %), and 22 (16.9 %) patients in the low, moderate, and high mucin groups, respectively. Patients with a high mucinous tumor component were significantly more likely to have an advanced tumor stage (p = 0.010) and a shorter disease-free (p = 0.002) and distant recurrence-free survivals (p < 0.001), whereas the mucinous tumor component showed no correlation with local recurrence (p = 0.101). A high mucinous component was also an independent predictive factor for a shorter disease-free survival (p = 0.041, hazard ratio = 2.56) and distant recurrence-free survival (p = 0.001, hazard ratio = 5.74) according to a multivariate analysis. CONCLUSIONS: Because the mucinous components showed little correlation with local recurrence, mucinous cancer should not be a determining factor for chemoradiotherapy. However, the frequent occurrence of metachronous distant metastasis among patients with a high mucin component makes this a possible indicator for more robust postoperative adjuvant treatment and close surveillance of recurrence.

cDNA cloning and characterization of vanadium-dependent bromoperoxidases from the red alga Laurencia nipponica.

The marine red alga genus Laurencia is one of the richest producers of unique brominated compounds in the marine environment. The cDNAs for two Laurencia nipponica vanadium-dependent bromoperoxidases (LnVBPO1 and LnVBPO2) were cloned and expressed in Escherichia coli. Enzyme assays of recombinant LnVBPO1 and LnVBPO2 using monochlorodimedone revealed that they were thermolabile but their Km values for Br(-) were significantly lower than other red algal VBPOs. The bromination reaction was also assessed using laurediol, the predicted natural precursor of the brominated ether laurencin. Laurediol, protected by trimethylsilyl at the enyne, was converted to deacetyllaurencin by the LnVBPOs, which was confirmed by tandem mass spectrometry. Native LnVBPO partially purified from algal bodies was active, suggesting that LnVBPO is functional in vivo. These results contributed to our knowledge of the biosynthesis of Laurencia brominated metabolites.

Tumescenamide C, a cyclic lipodepsipeptide from Streptomyces sp. KUSC_F05, exerts antimicrobial activity against the scab-forming actinomycete Streptomyces scabiei.

The antimicrobial activity of tumescenamide C against the scab-forming S. scabiei NBRC13768 was confirmed with a potent IC50 value (1.5 µg/mL). Three tumescenamide C-resistant S. scabiei strains were generated to compare their gene variants. All three resistant strains contained nonsynonymous variants in genes related to cellobiose/cellotriose transport system components; cebF1, cebF2, and cebG2, which are responsible for the production of the phytotoxin thaxtomin A. Decrease in thaxtomin A production and the virulence of the three resistant strains were revealed by the LC/MS analysis and necrosis assay, respectively. Although the nonsynonymous variants were insufficient for identifying the molecular target of tumescenamide C, the cell wall component wall teichoic acid (WTA) was observed to bind significantly to tumescenamide C. Moreover, changes in the WTA contents were detected in the tumescenamide C-resistant strains. These results imply that tumescenamide C targets the cell wall system to exert antimicrobial effects on S. scabiei.

Laparoscopy-Assisted Restorative Proctocolectomy with Ileal Pouch-Anal Anastomosis in Middle Colic Artery Ligation Immediately before Specimen Removal.

Introduction: Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) is the surgical procedure for ulcerative colitis (UC). Intestinal ischemia may occur if the main blood vessels are ligated at an early stage of this surgery. Considering that the blood flow in the large intestine can be maintained by preserving the middle colic artery, we have used a new IPAA method: ligating the middle colic artery immediately before removal of the specimens ("M-method"). Here, we evaluated the M-method's clinical outcomes. Methods: Between April 2009 and December 2021, 13 patients underwent a laparoscopy-assisted IPAA procedure at our institution. The conventional method was used for 6 patients, and the M-method was used for the other 7 patients. We retrospectively analyzed the cases' clinical notes. Results: The M-method's rate of postoperative complications (Clavien-Dindo classification grade II or more) was significantly lower than that of the conventional method (14.2% vs. 83.3%). The M-method group's postoperative stay period was also significantly shorter (average 16.4 days vs. 55.5). There were significant differences in the albumin value and the ratio of the modified GPS score 1 or 2 on the 7th postoperative day between the M- and conventional methods (average 3.15 vs. 2.5, average 4/7 vs. 6/6). However, it is necessary to consider the small number of cases and the uncontrolled historical comparison. Conclusion: Late ligation of the middle colic artery may be beneficial for patients' post-surgery recovery and can be recommended for IPAAs in UC patients.

The first case of laparoscopic surgery for cecal cancer occurring in a blind loop.

Cancer occurrence in a blind loop is extremely rare. An 86-year-old Japanese woman underwent colonoscopy for tarry stools and weight loss; it revealed a bypass of the transverse colon and small intestine, cecal cancer, and a polyp. She had suffered from acute appendicitis and had undergone two surgeries at age 25: an appendectomy and then a bypass surgery between the transverse colon and the small intestine. We performed a laparoscopy-assisted ileocecal resection for the cancer and polyp in the blind loop with an end-to-side instrumental anastomosis. The pathological examination demonstrated that the cancer was medullary carcinoma (T2, N0, M0, Stage I) and the polyp was tubular adenoma. Two months have passed since the patient's discharge, and she is free of abdominal complaints. Our literature search identified 10 cases of cancer in a blind loop. Laparoscopy-assisted surgery may be possible in patients who have undergone blind-loop surgery.

Prognostic Value of KRAS Exon-specific Mutations in Patients With Colorectal Cancer.

BACKGROUND/AIM: The clinical significance of many RAS-family mutations in colorectal cancer (CRC) remains unclear. The purpose of this study was to investigate the relationship of RAS mutations on an exon basis (i.e., mutations in KRAS exons 2, 3, and 4 and in NRAS) with clinicopathological features and prognosis in CRC. PATIENTS AND METHODS: We performed a retrospective cohort study of the medical records and frozen tissue samples of 268 consecutive patients with stage I-III CRC who underwent curative resection at a single institution between 2014 and 2018. RESULTS: The RAS mutation rate was significantly associated with age and histology. Patients with KRAS exon 2 mutations exhibited shorter recurrence-free survival compared to those with KRAS wild-type, KRAS exon 3 mutations, KRAS exon 4 mutations, and NRAS mutations (73.0% vs. 85.5%, 86.7%, 85.7%; p=0.031). Age and histology were independent risk factors for RAS mutations. RAS mutations were independent prognostic factors with respect to recurrence-free survival in patients with stage I-III CRC. CONCLUSION: In stage I-III CRC patients, KRAS exon 2 mutations had the worst prognosis, whereas KRAS wild type, exon 3 mutations, exon 4 mutations, and NRAS mutations had better prognoses.

Steroidogenic and maturation-inducing potency of native gonadotropic hormones in female chub mackerel, Scomber japonicus.

BACKGROUND: The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced in the pituitary gland and regulates gametogenesis through production of gonadal steroids. However, respective roles of two GtHs in the teleosts are still incompletely characterized due to technical difficulties in the purification of native GtHs. METHODS: Native FSH and LH were purified from the pituitaries of adult chub mackerel, Scomber japonicus by anion-exchange chromatography and immunoblotting using specific antisera. The steroidogenic potency of the intact chub mackerel FSH (cmFSH) and LH (cmLH) were evaluated in mid- and late-vitellogenic stage follicles by measuring the level of gonadal steroids, estradiol-17beta (Ε2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In addition, we evaluated the maturation-inducing potency of the GtHs on same stage follicles. RESULTS: Both cmFSH and cmLH significantly stimulated E2 production in mid-vitellogenic stage follicles. In contrast, only LH significantly stimulated the production of 17,20beta-P in late-vitellogenic stage follicles. Similarly, cmLH induced final oocyte maturation (FOM) in late-vitellogenic stage follicles. CONCLUSIONS: Present results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel.

Changes in the expression of pituitary gonadotropin subunits during reproductive cycle of multiple spawning female chub mackerel Scomber japonicus.

The endocrine regulation of reproduction in a multiple spawning fish with an asynchronous-type ovary remains largely unknown. The objectives of this study were to monitor changes in the mRNA expression of three gonadotropin (GtH) subunits (GPα, FSHß, and LHß) during the reproductive cycle of the female chub mackerel Scomber japonicus. Cloning and subsequent sequence analysis revealed that the cDNAs of chub mackerel GPα, FSHß, and LHß were 658, 535, and 599 nucleotides in length and encoded 117, 115, and 147 amino acids, respectively. We applied a quantitative real-time PCR assay to quantify the mRNA expression levels of these GtH subunits. During the seasonal reproductive cycle, FSHß mRNA levels remained high during the vitellogenic stages, while GPα and LHß mRNA levels peaked at the end of vitellogenesis. The expression of all three GtH subunits decreased during the post-spawning period. These results suggest that follicle-stimulating hormone (FSH) is involved in vitellogenesis, while luteinizing hormone (LH) functions during final oocyte maturation (FOM). Both GPα and FSHß mRNA levels remained high during the FOM stages of the spawning cycle and increased further just after spawning. Thus, FSH synthesis may be strongly activated just after spawning to accelerate vitellogenesis in preparation for the next spawning. Alternatively, LHß mRNA levels declined during hydration and then increased after ovulation. This study demonstrates that chub mackerel are a good model for investigating GtH functions in multiple spawning fish.

A Non-Invasive Deep Photoablation Technique to Inhibit DCIS Progression and Induce Antitumor Immunity.

Ductal carcinoma in situ (DCIS) of the breast is often managed by lumpectomy and radiation or mastectomy, despite its indolent features. Effective non-invasive treatment strategies could reduce the morbidity of DCIS treatment. We have exploited the high heat shock protein 90 (HSP90) activity in premalignant and malignant breast disease to non-invasively detect and selectively ablate tumors using photodynamic therapy (PDT). PDT with the HSP90-targeting photosensitizer, HS201, can not only ablate invasive breast cancers (BCs) while sparing non-tumor tissue, but also induce antitumor immunity. We hypothesized that HS201-PDT would both non-invasively ablate DCIS and prevent progression to invasive BC. We tested in vitro selective uptake and photosensitivity of HS201 in DCIS cell lines compared to the non-selective parental verteporfin, and assessed in vivo antitumor efficacy in mammary fat pad and intraductal implantation models. Selective uptake of HS201 enabled treatment of intraductal lesions while minimizing toxicity to non-tumor tissue. The in vivo activity of HS201-PDT was also tested in female MMTV-neu mice prior to the development of spontaneous invasive BC. Mice aged 5 months were administered HS201, and their mammary glands were exposed to laser light. HS201-PDT delayed the emergence of invasive BC, significantly prolonged disease-free survival (DFS) (p = 0.0328) and tended to improve overall survival compared to the no-treatment control (p = 0.0872). Systemic administration of anti-PD-L1 was combined with HS201-PDT and was tested in a more aggressive spontaneous tumor model, HER2delta16 transgenic mice. A single PDT dose combined with anti-PD-L1 improved DFS compared to the no-treatment control, which was significantly improved with repetitive HS201-PDT given with anti-PD-L1 (p = 0.0319). In conclusion, a non-invasive, skin- and tissue-sparing PDT strategy in combination with anti-PD-L1 antibodies effectively prevented malignant progression of DCIS to invasive BC. This non-invasive treatment strategy of DCIS may be safe and effective, while providing an option to reduce the morbidity of current conventional treatment for patients with DCIS. Clinical testing of HS201 is currently underway.

Combination of a novel heat shock protein 90-targeted photodynamic therapy with PD-1/PD-L1 blockade induces potent systemic antitumor efficacy and abscopal effect against breast cancers.

BACKGROUND: We previously demonstrated potent antitumor activity against human breast cancer xenografts using photodynamic therapy (PDT) targeting a novel tumor-specific photosensitizer (HS201), which binds heat shock protein 90 (HS201-PDT). However, induction of systemic antitumor immunity by HS201-PDT alone or by the combination strategy with immune checkpoint blockade has yet to be determined. METHODS: Using unilateral and bilateral implantation models of syngeneic breast tumors (E0771, MM3MG-HER2, and JC-HER3) in mice, we assessed whether HS201-PDT could induce local and systemic antitumor immunity. In an attempt to achieve a stronger abscopal effect for distant tumors, the combination strategy with anti-PD-L1 antibody was tested. Tumor-infiltrating leukocytes were analyzed by single cell RNA-sequencing and receptor-ligand interactome analysis to characterize in more detailed the mechanisms of action of the treatment and key signaling pathways involved. RESULTS: HS201-PDT demonstrated greater tumor control and survival in immune competent mice than in immunocompromised mice, suggesting the role of induced antitumor immunity; however, survival was modest and an abscopal effect on distant implanted tumor was weak. A combination of HS201-PDT with anti-PD-L1 antibody demonstrated the greatest antigen-specific immune response, tumor growth suppression, prolonged mouse survival time and abscopal effect. The most significant increase of intratumoral, activated CD8+T cells and decrease of exhausted CD8+T cells occurred following combination treatment compared with HS201-PDT monotherapy. Receptor-ligand interactome analysis showed marked enhancement of several pathways, such as CXCL, GALECTIN, GITRL, PECAM1 and NOTCH, associated with CD8+T cell activation in the combination group. Notably, the expression of the CXCR3 gene signature was the highest in the combination group, possibly explaining the enhanced tumor infiltration by T cells. CONCLUSIONS: The increased antitumor activity and upregulated CXCR3 gene signature induced by the combination of anti-PD-L1 antibody with HS201-PDT warrants the clinical testing of HS201-PDT combined with PD-1/PD-L1 blockade in patients with breast cancer, and the use of the CXCR3 gene signature as a biomarker.

Combination of ultrasound-based mechanical disruption of tumor with immune checkpoint blockade modifies tumor microenvironment and augments systemic antitumor immunity.

BACKGROUND: Despite multimodal adjuvant management with radiotherapy, chemotherapy and hormonal therapies, most surgically resected primary breast cancers relapse or metastasize. A potential solution to late and distant recurrence is to augment systemic antitumor immunity, in part by appropriately presenting tumor antigens, but also by modulating the immunosuppressive tumor microenvironment (TME). We previously validated this concept in models of murine carcinoma treated with a novel predominately microcavitating version of high-intensity focused ultrasound (HIFU), mechanical high-intensity focused ultrasound (M-HIFU). Here we elucidated the mechanisms of enhanced antitumor immunity by M-HIFU over conventional thermal high-intensity focused ultrasound (T-HIFU) and investigated the potential of the combinatorial strategy with an immune checkpoint inhibitor, anti-PD-L1 antibody. METHODS: The antitumor efficacy of treatments was investigated in syngeneic murine breast cancer models using triple-negative (E0771) or human ErbB-2 (HER2) expressing (MM3MG-HER2) tumors in C57BL/6 or BALB/c mice, respectively. Induction of systemic antitumor immunity by the treatments was tested using bilateral tumor implantation models. Flow cytometry, immunohistochemistry, and single-cell RNA sequencing were performed to elucidate detailed effects of HIFU treatments or combination treatment on TME, including the activation status of CD8 T cells and polarization of tumor-associated macrophages (TAMs). RESULTS: More potent systemic antitumor immunity and tumor growth suppression were induced by M-HIFU compared with T-HIFU. Molecular characterization of the TME after M-HIFU by single-cell RNA sequencing demonstrated repolarization of TAM to the immunostimulatory M1 subtype compared with TME post-T-HIFU. Concurrent anti-PD-L1 antibody administration or depletion of CD4+ T cells containing a population of regulatory T cells markedly increased T cell-mediated antitumor immunity and tumor growth suppression at distant, untreated tumor sites in M-HIFU treated mice compared with M-HIFU monotherapy. CD8 T and natural killer cells played major roles as effector cells in the combination treatment. CONCLUSIONS: Physical disruption of the TME by M-HIFU repolarizes TAM, enhances T-cell infiltration, and, when combined with anti-PD-L1 antibody, mediates superior systemic antitumor immune responses and distant tumor growth suppression. These findings suggest M-HIFU combined with anti-PD-L1 may be useful in reducing late recurrence or metastasis when applied to primary tumors.

HSP90-Specific nIR Probe Identifies Aggressive Prostate Cancers: Translation from Preclinical Models to a Human Phase I Study.

A noninvasive test to discriminate indolent prostate cancers from lethal ones would focus treatment where necessary while reducing overtreatment. We exploited the known activity of heat shock protein 90 (Hsp90) as a chaperone critical for the function of numerous oncogenic drivers, including the androgen receptor and its variants, to detect aggressive prostate cancer. We linked a near-infrared fluorescing molecule to an HSP90 binding drug and demonstrated that this probe (designated HS196) was highly sensitive and specific for detecting implanted prostate cancer cell lines with greater uptake by more aggressive subtypes. In a phase I human study, systemically administered HS196 could be detected in malignant nodules within prostatectomy specimens. Single-cell RNA sequencing identified uptake of HS196 by malignant prostate epithelium from the peripheral zone (AMACR+ERG+EPCAM+ cells), including SYP+ neuroendocrine cells that are associated with therapeutic resistance and metastatic progression. A theranostic version of this molecule is under clinical testing.

Heterogenous chemosensitivity of a panel of organoid lines derived from small cell neuroendocrine carcinoma of the uterine cervix.

Small cell neuroendocrine carcinoma (SCNEC) of the uterine cervix is a rare disease with a poor prognosis. The lack of established disease models has hampered therapy development. We generated a panel of cancer tissue-originated spheroid (CTOS) lines derived from SCNEC of the uterine cervix using a method based upon cell-cell contact throughout the preparation and culturing processes. Using 11 CTOS lines, we assessed the sensitivity of various drugs used in clinical practice. Drug sensitivity assays revealed significant heterogeneous inter-CTOS chemosensitivity. Microarray analyses were then performed to identify sensitivity-related gene signatures. Specific gene sets were identified which likely contribute to the sensitivity to the tested drugs. We identified a line (Cerv54) that was exceptionally sensitive to irinotecan. Cerv54 had increased levels of CES1, which catalyzes the conversion of irinotecan to the active form, SN38, although in Cerv54 cells, SN38 was undetectable, CES1 expression and activity were markedly low compared to the liver, and a CES1 inhibitor had no effect on irinotecan sensitivity. These results suggested a novel irinotecan mode of action in Cerv54. Our CTOS lines may be useful for understanding the variation and mechanism of drug sensitivity, contributing to the understanding and development of chemotherapeutic drugs.

Heat shock protein 90-targeted photodynamic therapy enables treatment of subcutaneous and visceral tumors.

Photodynamic therapy (PDT) ablates malignancies by applying focused near-infrared (nIR) light onto a lesion of interest after systemic administration of a photosensitizer (PS); however, the accumulation of existing PS is not tumor-exclusive. We developed a tumor-localizing strategy for PDT, exploiting the high expression of heat shock protein 90 (Hsp90) in cancer cells to retain high concentrations of PS by tethering a small molecule Hsp90 inhibitor to a PS (verteporfin, VP) to create an Hsp90-targeted PS (HS201). HS201 accumulates to a greater extent than VP in breast cancer cells both in vitro and in vivo, resulting in increased treatment efficacy of HS201-PDT in various human breast cancer xenografts regardless of molecular and clinical subtypes. The therapeutic index achieved with Hsp90-targeted PDT would permit treatment not only of localized tumors, but also more diffusely infiltrating processes such as inflammatory breast cancer.

Methylation deficiency disrupts biological rhythms from bacteria to humans.

The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies.