Prediction of cell cycle distribution after drug exposure by high content imaging analysis using low-toxic DNA staining dye.

Interference in cell cycle progression has been noted as one of the important properties of anticancer drugs. In this study, we developed the cell cycle prediction model using high-content imaging data of recipient cells after drug exposure and DNA-staining with a low-toxic DNA dye, SiR-DNA. For this purpose, we exploited HeLa and MCF7 cells introduced with a fluorescent ubiquitination-based cell cycle indicator (Fucci). Fucci-expressing cancer cells were subjected to high-content imaging analysis using OperettaCLS after 36-h exposure to anticancer drugs; the nuclei were segmented, and the morphological and intensity properties of each nucleus characterized by SiR-DNA staining were calculated using imaging analysis software, Harmony. For the use of training, we classified cells into each phase of the cell cycle using the Fucci system. Training data (n = 7500) and validation data (n = 2500) were randomly sampled and the binary classification prediction models for G1, early S, and S/G2/M phases of the cell cycle were developed using four supervised machine learning algorithms. We selected random forest as the model with the best performance through 10-fold cross-validation; the accuracy rate was approximately 75%-87%. Regarding feature importance, variables expected to be biologically related to the cell cycle, for example, signal intensity and nuclear size, were highly ranked, suggesting the validity of the model. These results showed that the cell cycle can be predicted in cancer cells by simply exploiting the current prediction model using fluorescent images of DNA-staining dye, and the model could be applied for the use of future ex vivo drug sensitivity diagnosis.

[Questionnaire Survey on the Timing of Comprehensive Genomic Profiling for Advanced Recurrent Colorectal Cancer and Its Impact on Patients].

BACKGROUNDS: Comprehensive genomic profiling(CGP)has been covered by health insurance since June 2019. However, the clinical impact of CGP on patients with metastatic colorectal cancer(mCRC)remains unclear. To date, there are very limited reports regarding patient-oriented outcomes of CGP in mCRC. PATIENTS: A questionnaire was completed by patients with mCRC who had already received their CGP results after April 2021. Eight questions were posed, covering the degree of satisfaction and timing when CGP was conducted. RESULTS: Of the 51 patients with mCRC who had received their CGP test results by August 2021 in our department, 21 patients responded to our questionnaire. In total 66.7% patients with mCRC answered "(very)satisfied"with the CGP testing. 28.6% of the patients already knew about CGP testing before their local doctors informed them. Except for 3 patients who did not answer, 47.6% and 9.5% of patients with mCRC"agreed"and "moderately agreed"with the timing of the CGP test. CONCLUSION: Although most patients with mCRC failed to access promising new treatment via CGP, approximately half of the patients answered that they were satisfied with the CGP testing. Conversely, a few patients already knew about CGP testing before it was proposed by their doctors. Thus, the provision of information at an early stage is necessary.

[Educational Project of Self-Removal of the Intravenous Central Venous Access Port Guided by Patients with Advanced Colorectal Cancer Instructed by a Nurse in the University Hospital].

BACKGROUND: Fluorouracil infusion for 46±5h from the central venous(CV)port is required for mFOLFOX6, FOLFIRI, and FOLFOXIRI in patients with advanced colorectal cancer(CRC), followed by self-removal of the needle by patients. At our hospital, outpatients were instructed for self-removal of the needle, but the results were unsatisfactory. Therefore, instructions for self-removal of the needle from the CV port have been initiated at the patient ward since April 2019, making use of a hospital stay of 3 days. PATIENTS: We retrospectively enrolled patients with chemotherapy-introduced advanced CRC from the CV port who received instructions for self-removal of the needle in the outpatient department and ward between January 2018 and December 2021. RESULTS: Of all patients with advanced CRC, 21 received instructions at the outpatient department(OP)while 67 at patient ward(PW). Incidences of successful self-removal of the needle without the aid of others were similar: 47% in OP and 52% in PW(p=0.80). However, after several additional instructions involving their families, it was higher in PW than in OP(97.0 vs 76.1%, p=0.005). Incidences of successful self-removal of the needle without the aid of others in those aged≥75/<75, and≥65/<65 years were 0%/61.1%, and 35.4%/67.5%, respectively. OP was as a risk factor for failed self-removal of the needle in the logistic regression analysis(odds ratio: 11.19, 95%CI: 1.86- 67.30). CONCLUSION: Repeated instructions involving patients' families during the hospital stay improved the incidence of successful self-removal of the needle. Involvement of patients' families from the beginning may effectively improve self- removal of the needle, particularly in the elderly patients with advanced CRC.

Comprehensive Genomic Profiling Detects Hereditary Cancers and Confers Survival Advantage in Patients With Gynaecological Cancers.

BACKGROUND/AIM: The clinical benefits of comprehensive genomic profiling (CGP) of tumours in patients with gynaecological cancers remain unknown. We investigated the utility of CGP in assessing patient survival and its efficacy in detecting hereditary cancers in gynaecological patients. PATIENTS AND METHODS: We retrospectively analysed the medical records of 104 gynaecological patients who underwent CGP between August 2018 and December 2022. The detection of actionable and accessible genomic alterations and administration of targeted therapy, as recommended by the molecular tumour board (MTB), were assessed. The overall survival (after second-line treatment in cervical and endometrial carcinomas and after platinum-resistant recurrence in ovarian carcinoma) was compared between patients with or without administration of MTB-recommended genotype-matched therapy. Germline findings were assessed using a variant allele frequency-tumour content graph. RESULTS: Among 104 patients, actionable and accessible genomic alterations were observed in 53 patients. Matched therapy was applied in 21 patients, comprising administration of repurposing itraconazole (n=7), immune checkpoint inhibitors (n=7), poly (ADP-ribose) polymerase inhibitors (n=5), and others (n=2). The median overall survival of patients receiving and not receiving matched therapy were 19.3 months and 11.2 months, respectively (p=0.036, hazard ratio=0.48). Among 12 patients with hereditary cancers, 11 patients were previously undiagnosed. Seven patients had hereditary breast and ovarian cancer, and five had other cancer. CONCLUSION: The implementation of CGP testing prolonged overall survival in gynaecological cancer as well as provided an opportunity for genetic counselling for newly-diagnosed patients with hereditary cancers and their families.

[Challenge of Skin Treatment Led by Nurses and Pharmacists for Advanced Colorectal Cancer Patients Treated with Anti-EGFR Antibodies].

Preemptive skin treatment led by nurses and pharmacists was started for patients with metastatic colorectal cancer (mCRC)who received anti-EGFR antibody treatment. Incidence of skin-related toxicities, amount of topical moisturizers used, and administered cycles of anti-EGFR antibody were retrospectively compared between a preemptive skin treatment group and a control group. Thirty-four mCRC patients before the introduction of preemptive skin treatment led by nurses and 23 mCRC patients treated with preemptive skin treatment led by nurses were evaluated. The incidence of 6- and 12- week Grade 2 or higher skin-related toxicity was 23.5% in the control group and 8.7% in the preemptive group(p=0.18), and 67.7% in the control group and 30.4% in the preemptive group(p=0.0076), respectively. Mean amounts of moisturizer used were both lower in the control group than in the preemptive group at both 6 weeks and 7-12 weeks(6 weeks; 275 g vs 550 g, p=0.036, 7-12 weeks; 575 g vs 1,175 g, p=0.013). However, the amount of topical steroid used was similar in both groups. Preemptive moisturizer skin treatment led by nurses and pharmacists may decrease the incidence of skin- related toxicity.

[Cross-Functional Project of Using Information-Sharing Tool for Metastatic Colorectal Cancer Patients in University Hospital].

Treatment for late-stage cancer patients should be discussed depending on the patients' will, however it is not sometimes fully discussed in our daily practice. Based on this background, the information-sharing tool for metastatic colorectal cancer patients, who are refractory to first-line and second-line chemotherapy and/or who are given a year to live, has been introduced in our university hospital since November 2019. To evaluate the utility of this tool, the influence of the tools on the outcome of the patients was evaluated. Regarding the comparison between the patients before and after the introduction of the information-sharing tool, the period between the day of the consent to the DNAR between the day of the death is longer in the patients after the introduction than those before the introduction(median 43 vs 6 days, p=0.025). The introduction of the information-sharing tool can provide the metastatic colorectal cancer patients with more opportunities to discuss how to spend the rest of their lives and with longer time at the place where they want to stay.

Mechanistic study of PpIX accumulation using the JFCR39 cell panel revealed a role for dynamin 2-mediated exocytosis.

5-aminolevulinic acid (5-ALA) has recently been employed for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various types of cancer because hyperproliferating tumor cells do not utilize oxidative phosphorylation and do not efficiently produce heme; instead, they accumulate protoporphyrin IX (PpIX), which is a precursor of heme that is activated by violet light irradiation that results in the production of red fluorescence and singlet oxygen. The efficiencies of ALA-PDD and ALA-PDT depend on the efficient cellular uptake of 5-ALA and the inefficient excretion of PpIX. We employed the JFCR39 cell panel to determine whether tumor cells originating from different tissues can produce and accumulate PpIX. We also investigated cellular factors/molecules involved in PpIX excretion by tumor cells with the JFCR39 cell panel. Unexpectedly, the expression levels of ABCG2, which has been considered to play a major role in PpIX extracellular transport, did not show a strong correlation with PpIX excretion levels in the JFCR39 cell panel, although an ABCG2 inhibitor significantly increased intracellular PpIX accumulation in several tumor cell lines. In contrast, the expression levels of dynamin 2, which is a cell membrane-associated molecule involved in exocytosis, were correlated with the PpIX excretion levels. Moreover, inhibitors of dynamin significantly suppressed PpIX excretion and increased the intracellular levels of PpIX. This is the first report demonstrating the causal relationship between dynamin 2 expression and PpIX excretion in tumor cells.

Antitumor profile of the PI3K inhibitor ZSTK474 in human sarcoma cell lines.

Treatment of patients with advanced sarcoma remains challenging due to lack of effective medicine, with the development of novel drugs being of keen interest. A pan-PI3K inhibitor, ZSTK474, has been evaluated in clinical trials against a range of advanced solid tumors, with clinical benefit shown in sarcoma patients. In the present study, we developed a panel of 14 human sarcoma cell lines and investigated the antitumor effect of 24 anticancer agents including ZSTK474, other PI3K inhibitors, and those clinically used for sarcoma treatment. ZSTK474 exhibited a similar antiproliferative profile to other PI3K inhibitors but was clearly different from the other drugs examined. Indeed, ZSTK474 inhibited PI3K-downstream pathways, in parallel to growth inhibition, in all cell lines examined, showing proof-of-concept of PI3K inhibition. In addition, ZSTK474 induced apoptosis selectively in Ewing's sarcoma (RD-ES and A673), alveolar rhabdomyosarcoma (SJCRH30) and synovial sarcoma (SYO-1, Aska-SS and Yamato-SS) cell lines, all of which harbor chromosomal translocation and resulting oncogenic fusion genes, EWSR1-FLI1, PAX3-FOXO1 and SS18-SSX, respectively. Finally, animal experiments confirmed the antitumor activity of ZSTK474 in vivo, with superior efficacy observed in translocation-positive cells. These results suggest that ZSTK474 could be a promising drug candidate for treating sarcomas, especially those harboring chromosomal translocation.

Basal expression of insulin-like growth factor 1 receptor determines intrinsic resistance of cancer cells to a phosphatidylinositol 3-kinase inhibitor ZSTK474.

Drug resistance often critically limits the efficacy of molecular targeted drugs. Although pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K) is an attractive therapeutic strategy for cancer therapy, molecular determinants for efficacy of PI3K inhibitors (PI3Kis) remain unclear. We previously identified that overexpression of insulin-like growth factor 1 receptor (IGF1R) contributed to the development of drug resistance after long-term exposure to PI3Kis. In this study, we examined the involvement of basal IGF1R expression in intrinsic resistance of drug-naïve cancer cells to PI3Kis and whether inhibition of IGF1R overcomes the resistance. We found that cancer cells highly expressing IGF1R showed resistance to dephosphorylation of Akt and subsequent antitumor effect by ZSTK474 treatment. Knockdown of IGF1R by siRNAs facilitated the dephosphorylation and enhanced the drug efficacy. These cells expressed tyrosine-phosphorylated insulin receptor substrate 1 at high levels, which was dependent on basal IGF1R expression. In these cells, the efficacy of ZSTK474 in vitro and in vivo was improved by its combination with the IGF1R inhibitor OSI-906. Finally, we found a significant correlation between the basal expression level of IGF1R and the inefficacy of ZSTK474 in an in vivo human cancer panel, as well as in vitro. These results suggest that basal IGF1R expression affects intrinsic resistance of cancer cells to ZSTK474, and IGF1R is a promising target to improve the therapeutic efficacy. The current results provide evidence of combination therapy of PI3Kis with IGF1R inhibitors for treating IGF1R-positive human cancers.

Establishment of phosphatidylinositol 3-kinase inhibitor-resistant cancer cell lines and therapeutic strategies for overcoming the resistance.

Acquired resistance is a major obstacle for conventional cancer chemotherapy, and also for some of the targeted therapies approved to date. Long-term treatment using protein tyrosine kinase inhibitors (TKIs), such as gefitinib and imatinib, gives rise to resistant cancer cells carrying a drug-resistant gatekeeper mutation in the kinase domain of the respective target genes, EGFR and BCR-ABL. As for the phosphatidylinositol 3-kinase inhibitors (PI3Kis), little is known about their acquired resistance, although some are undergoing clinical trials. To address this issue, we exposed 11 human cancer cell lines to ZSTK474, a PI3Ki we developed previously, for a period of more than 1 year in vitro. Consequently, we established ZSTK474-resistant cells from four of the 11 cancer cell lines tested. The acquired resistance was not only to ZSTK474 but also to other PI3Kis. None of the PI3Ki-resistant cells, however, contained any mutation in the kinase domain of the PIK3CA gene. Instead, we found that insulin-like growth factor 1 receptor (IGF1R) was overexpressed in all four resistant cells. Interestingly, targeted knockdown of IGF1R expression using specific siRNAs or inhibition of IGF1R using IGF1R-TKIs reversed the acquired PI3Ki resistance. These results suggest that long-term treatment with PI3Kis may cause acquired resistance, and targeting IGF1R is a promising strategy to overcome the resistance.

Cytometric analysis of cytotoxicity of polyphenols and related phenolics to rat thymocytes: potent cytotoxicity of resveratrol to normal cells.

The chemopreventive and chemotherapeutic actions of polyphenols and related phenolics have received considerable attention since these compounds induce apoptosis in several types of cancer cells in vitro. A plausible criterion for the use of such compounds is that they should not exert any toxic effect on normal cells. However, information about the toxicity of polyphenols and related phenolics to normal cells is limited. In this study, the effects of polyphenols and related phenolics on rat thymocytes were examined by flow cytometric analysis with appropriate fluorescent probes. The compounds examined in this study were caffeic acid, rosmarinic acid, chlorogenic acid, (+)-catechin, 6-gingerol, sesamol, resveratrol, and eugenol. Of these, resveratrol was the most cytotoxic on rat thymocytes incubated for 24 hrs with 100 microM of this compound. Resveratrol at a concentration of 10 microM or more (up to 100 microM) led to a significant dose-dependent increase in the population of dead cells, shrunken living cells, annexin V-positive cells and cells with hypodiploidal DNA. In the presence of benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK), a pan-inhibitor of caspases, the resveratrol-induced increase in the population of cells with hypodiploidal DNA was partially inhibited. Overall, it is suggested that resveratrol at a concentration of 10 microM or more induces apoptosis in normal cells as well as cancer cells (previously reported elsewhere). Thus, at concentrations that are suitable for chemopreventive and chemotherapeutic actions, resveratrol may exert a cytotoxic effect on normal cells.

Proteomics-based identification of biomarkers for predicting sensitivity to a PI3-kinase inhibitor in cancer.

To identify biomarkers for predicting sensitivity to phosphatidylinositol 3-kinase (PI3K) inhibitors, we have developed a proteomics-based approach. Using surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS), we measured the expression of 393 proteins in 39 human cancer cell lines (JFCR-39), and combined it with our previously established chemosensitivity database to select for proteins whose expressions show significant correlations to drug sensitivities. This integrated approach allowed us to identify peaks from two proteins, 11.6 and 11.8 kDa, that showed significant correlations with the sensitivity to a PI3K inhibitor, LY294002. We found that the 11.8 kDa protein was a phosphorylated form of the 11.6 kDa protein. While the 11.8 kDa protein showed a positive correlation with the sensitivity to LY294002, the 11.6 kDa protein showed a negative correlation with that of the LY294002. The 11.6 kDa protein was purified chromatographically, and was identified by SELDI-TOF MS as the ribosomal P2 protein, which possesses two prospective phosphorylation sites. These results suggested that the phosphorylation status of the ribosomal P2 was responsible for determining the sensitivity to LY294002, and that the ribosomal P2 could be a potential biomarker for predicting chemosensitivity.

Clotrimazole, an antifungal drug possessing diverse actions, increases the vulnerability to cadmium in lymphocytes dissociated from rat thymus.

Since clotrimazole, known as an antifungal drug, exerts diverse actions on cellular functions, it is expected that clotrimazole can be used for other purposes. This antifungal drug protects the cells overloaded with Ca(2+) by A23187, a calcium ionophore. Therefore, the agent may prevent the cells from death induced by heavy metals such as CdCl(2), PbCl(2), or HgCl(2) that are respectively proposed to increase intracellular Ca(2+) concentration. To test this possibility, we have examined the effect of clotrimazole on the cells simultaneously treated with CdCl(2), PbCl(2), or HgCl(2) using rat thymocytes and a flow cytometer with fluorescent probes. The simultaneous application of clotrimazole and CdCl(2) significantly decreased cell viability, even though the concentrations of both were ineffective at affecting the viability. The significant decrease in cell viability was not due to the inhibition of Ca(2+)-ATPase and Ca(2+)-dependent K(+) channels that were induced by clotrimazole. The simultaneous application increased the population of cells with phosphatidylserine exposed on membrane surface, indicating the change in asymmetrical distribution of membrane phospholipids. Furthermore, the cytotoxicity induced by the combination of clotrimazole and CdCl(2) under nominally Ca(2+)-free condition was more profound than that under normal Ca(2+) condition. Therefore, the membrane may be a target for the cytotoxic action of clotrimazole and CdCl(2) that were simultaneously applied. It is also the case for PbCl(2), but not the case for HgCl(2). It is concluded that clotrimazole can modulate the cytotoxicity of some heavy metals.

Increase in intracellular Cd(2+) concentration of rat cerebellar granule neurons incubated with cadmium chloride: cadmium cytotoxicity under external Ca(2+)-free condition.

In order to examine the cadmium cytotoxicity unrelated to external Ca(2+), the effects of micromolar CdCl(2) on intracellular Cd(2+) concentration, cellular content of glutathione, and cell viability of rat cerebellar granule neurons were examined under normal Ca(2+) and external Ca(2+)-free conditions, using a laser confocal microscope with fluorescent probes, fluo-3-AM, 5-chloromethylfluorescein (CMF) diacetate, and propidium iodide. CdCl(2) (10-300 microM) dose-dependently increased the intensity of fluo-3 fluorescence. Exposure to CdCl(2) equally enhanced the fluo-3 fluorescence under both Ca(2+) conditions and MnCl(2) did not quench the CdCl(2)-enhanced fluorescence. The results indicate that the enhancement of fluo-3 fluorescence is due to the increase in intracellular Cd(2+) concentration. CdCl(2) at 100-300 microM decreased the intensity of CMF fluorescence, indicating the decrease in cellular content of glutathione. The population of cells stained with propidium (dead cells) was increased by 100-300 microM CdCl(2). Similar results described above were also observed under external Ca(2+)-free condition. It is suggested that some of cytotoxic actions of CdCl(2) on neurons are unrelated to external Ca(2+), one of main sources for increasing intracellular Ca(2+) concentration.

Cremophor EL augments the cytotoxicity of hydrogen peroxide in lymphocytes dissociated from rat thymus glands.

The pharmaceutical uses of cremophor EL, a non-ionic surfactant, are similar to those of polysorbate 80. In our previous study, polysorbate 80 exerted some adverse actions on rat thymocytes under in vitro condition. Therefore, the effects of cremophor EL on thymic lymphocytes were examined using a flow cytometer with appropriate fluorescent dyes. Cremophor EL at 10 microg/ml or more (up to 300 microg/ml) concentration-dependently decreased cellular content of glutathione. The cell viability of thymocytes under control condition was 95.4 +/- 1.2% (n = 7, mean +/- S.D.). The incubation of thymocytes with 300 microg/ml cremophor EL or 3 mM hydrogen peroxide for 2 h, respectively, decreased the cell viability to 90.8 +/- 2.8% or 91.2 +/- 2.6%. However, the simultaneous incubation with cremophor EL and hydrogen peroxide decreased the cell viability to 28.7 +/- 8.2%. Cremophor EL at 100 microg/ml accelerated the process of cell death induced by hydrogen peroxide. Results suggest that cremophor EL increases the susceptibility to oxidative stress. Cremophor EL at clinically relevant concentrations may increase the therapeutic potential of some anticancer agents to produce oxidative stress.

Rapid discovery and identification of a tissue-specific tumor biomarker from 39 human cancer cell lines using the SELDI ProteinChip platform.

Useful biomarkers are needed for early detection of cancers. To demonstrate the potential diagnostic usefulness of a new proteomic technology, we performed Expression Difference Mapping analysis on 39 cancer cell lines from 9 different tissues using ProteinChip technology. A protein biomarker candidate of 12kDa was found in colon cancer cells. We then optimized the purification conditions for this biomarker by utilizing Retentate Chromatography mass spectrometry (RC-MS). The optimized purification conditions developed "on-chip" were directly transferred to conventional chromatography to purify the biomarker, which was identified as prothymosin-alpha by ProteinChip time-of-flight mass spectrometry (TOF MS) and ProteinChip-Tandem MS systems. The relative expression level of prothymosin-alpha between colon cancer cells and normal colon mucosal cells was evaluated on the same ProteinChip platform. Prothymosin-alpha expression in colon cancer cells was clearly higher than in normal colon cells. These results indicate that prothymosin-alpha could be a potential biomarker for colon cancer, and that the ProteinChip platform could perform the whole process of biomarker discovery from screening to evaluation of the identified marker.