Natural Compound-Generated Oxidative Stress: From Bench to Bedside

Oxidants are constantly generated in a biological system as a result of physiological processes. However, an imbalance between oxidants and antioxidants can lead to a pathophysiological condition known as oxidative stress. Natural compounds as inducers of oxidative stress are able to modulate physiological functions of cancer cells leading to cell death or survival. This chapter aims at providing an overview of pro- and antioxidant activities of natural compounds related to cancer and related therapies

Raloxifene along with curcumin analogue RL91, a combinational approach against hormone refractory prostate cancer

Background: Approximately 80% of prostate cancer patients undergoing hormone therapy progress within 12-18 months to a hormone insensitive form of the disease known as hormone refractory prostate cancer. The median survival rate is less than 12 months and traditional therapeutic agents have shown little effect on the progression of the disease. The selective estrogen receptor modulator (SERM), raloxifene has been examined in a limited phase ІІ clinical trial and showed anti-tumor properties. The current study investigated the activity of raloxifene on expression of ERα, ERβ, AR, EGFR and caveolin-1. Also the study aimed to see whether synergistic cytotoxicity could be obtained administering raloxifene in combination with the curcumin analogue RL91. Methods: Two different hormone refractory prostate cancer cell lines PC3 and DU-145 were used in the study. PC3 cells were specifically used to understand the role of raloxifene 10 and 15 μM on different receptors such as estrogen receptor α (ERα), estrogen receptor β (ERβ), androgen receptor (AR), epidermal growth factor receptor (EGFR) and caveolin-1. Since raloxifene is a SERM it should primarily target estrogen receptors. We wanted to determine the effect of raloxifene on different receptors. Immunocytochemistry and Western blotting was carried out to analyze the change in receptor localization and quantitative protein expression levels, respectively. To assess the synergistic potential of the combinational therapy, raloxifene 5, 10 μM and RL91 1.5 and 2 μM were measured individually and in combination on PC3 and DU-145 using sulforhodamine B assay. Results: Raloxifene treatment affected the localization of ERβ, EGFR, AR and caveolin-1 in PC3 cells. The drug was shown to increase the translocation of ERβ and EGFR after 6 h of treatment with a maximum difference observed after 48 h. Also, a decreased expression of ERβ and EGFR was shown following 48 h of raloxifene treatment. It also decreased the co-localization of the EGFR and caveolin-1. Apart from this, it also induced vesicle formation and accumulation of cellular content inside the cells. The cytotoxicity assay showed more cytotoxic potential of RL91 compared to raloxifene, however the combination showed synergism with more than 80 % of cell death observed for both PC3 and DU-145 cells following raloxifene 10 μM and RL91 1.5 μM treatment. Conclusions: In HRPC, ER and EGFR mediated signaling is important for the proliferation of the cells. Raloxifene’s modulation of ERβ, EGFR and AR develops a stressed condition for the cells which decreased the proliferation. By this mechanism raloxifene enhances the cytotoxicity elicited by RL91 when administered in combination

Raloxifene along with curcumin analogue RL91, a combinational approach against hormone refractory prostate cancer

Background: Approximately 80% of prostate cancer patients undergoing hormone therapy progress within 12-18 months to a hormone insensitive form of the disease known as hormone refractory prostate cancer. The median survival rate is less than 12 months and traditional therapeutic agents have shown little effect on the progression of the disease. The selective estrogen receptor modulator (SERM), raloxifene has been examined in a limited phase ІІ clinical trial and showed anti-tumor properties. The current study investigated the activity of raloxifene on expression of ERα, ERβ, AR, EGFR and caveolin-1. Also the study aimed to see whether synergistic cytotoxicity could be obtained administering raloxifene in combination with the curcumin analogue RL91. Methods: Two different hormone refractory prostate cancer cell lines PC3 and DU-145 were used in the study. PC3 cells were specifically used to understand the role of raloxifene 10 and 15 μM on different receptors such as estrogen receptor α (ERα), estrogen receptor β (ERβ), androgen receptor (AR), epidermal growth factor receptor (EGFR) and caveolin-1. Since raloxifene is a SERM it should primarily target estrogen receptors. We wanted to determine the effect of raloxifene on different receptors. Immunocytochemistry and Western blotting was carried out to analyze the change in receptor localization and quantitative protein expression levels, respectively. To assess the synergistic potential of the combinational therapy, raloxifene 5, 10 μM and RL91 1.5 and 2 μM were measured individually and in combination on PC3 and DU-145 using sulforhodamine B assay. Results: Raloxifene treatment affected the localization of ERβ, EGFR, AR and caveolin-1 in PC3 cells. The drug was shown to increase the translocation of ERβ and EGFR after 6 h of treatment with a maximum difference observed after 48 h. Also, a decreased expression of ERβ and EGFR was shown following 48 h of raloxifene treatment. It also decreased the co-localization of the EGFR and caveolin-1. Apart from this, it also induced vesicle formation and accumulation of cellular content inside the cells. The cytotoxicity assay showed more cytotoxic potential of RL91 compared to raloxifene, however the combination showed synergism with more than 80 % of cell death observed for both PC3 and DU-145 cells following raloxifene 10 μM and RL91 1.5 μM treatment. Conclusions: In HRPC, ER and EGFR mediated signaling is important for the proliferation of the cells. Raloxifene’s modulation of ERβ, EGFR and AR develops a stressed condition for the cells which decreased the proliferation. By this mechanism raloxifene enhances the cytotoxicity elicited by RL91 when administered in combination

Raloxifene Suppresses Tumor Growth and Metastasis in an Orthotopic Model of Castration-Resistant Prostate Cancer

Androgen receptor (AR)-castrate-resistant prostate cancer (CRPC) is an aggressive form of prostate cancer that does not have clinically approved targeted treatment options. To this end, the cytotoxic potential of raloxifene and the synthetic curcumin derivative 2,6-bis (pyridin-4-ylmethylene)-cyclohexanone (RL91) was examined in AR-(PC3 and DU145) cells and AR+ (LnCaP) CRPC cells. The results showed that both raloxifene and RL91 elicited significant cytotoxicity across three cell lines with the lowest EC50 values in PC3 cells. Additionally, the two drugs were synergistically cytotoxic toward the PC3, DU-145 and LNCaP cell lines. To determine the effect of the drug combination in vivo, an orthotopic model of CRPC was used. Male mice were injected with PC3 prostate cancer cells and then treated with vehicle (5 mL/kg), raloxifene (8.5 mg/kg, po), RL91 (8.5 mg/kg, po) or a combination of raloxifene and RL91 for six weeks. Sham animals were subjected to the surgical procedure but were not implanted with PC3 cells. The results showed that raloxifene decreased tumor size and weight as well as metastasis to renal lymph nodes. However, combination treatment reversed the efficacy of raloxifene as tumor volume and metastasis returned to control levels. The results suggest that raloxifene has tumor suppressive and anti-metastatic effects and has potential for further clinical use in AR-CRPC

Abstract 2521: Race specific differences in DNA damage repair dysregulation in breast cancer and association with outcome

Abstract African American (AA) estrogen receptor positive (ER+) breast cancer patients have worse outcomes than Caucasian Americans (CA) being 42 % more likely to die from the disease. However, AAs are severely underrepresented in currently available datasets of patient tumors, which precludes comprehensive approaches to identify race-specific molecular drivers of these poor outcomes. Endocrine therapy (ET) is the first line standard of care for ER+ breast cancer. Nevertheless, 1 in 4 patients develop ET resistance. Specific DNA damage/repair (DDR) defects have been shown to associate with poor outcome in CA patients, and to induce ET resistance. Whether these or other DDR defects contribute to poor outcomes observed in AA patients remains unknown. For the purpose of this investigation, we assessed the DDR dysregulation landscape of AA ER+ tumors using three independent tumor datasets (1) GSE78958 (2) GSE18229 (3) The Cancer Genome Atlas (TCGA) and two normal breast datasets (1) GSE43973 (2) GSE50939. This analysis identified a distinct set of AA ER+ tumors with simultaneous dysregulation of genes from multiple DDR pathways, rarely seen in CA tumors. This simultaneous dysregulation also associated with worse patient outcomes in all three datasets analyzed. This work constitutes the first systematic analysis of race-specific DDR dysregulation in ER+ breast cancer, and identifies DDR as a potential predictive biomarker for worse outcome seen in AA patients. Citation Format: Aloran Mazumder, Athena Jimenez, Rachel Ellsworth, Stephen Freedland, Sophia George, Matthew Bainbridge, Svasti Haricharan. Race specific differences in DNA damage repair dysregulation in breast cancer and association with outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2521

Human telomerase reverse transcriptase depletion potentiates the growth- T inhibitory activity of imatinib in chronic myeloid leukemia stem cells

Although tyrosine kinase inhibitors (TKIs) revolutionized the management of chronic myeloid leukemia (CML), resistance against TKIs and leukemia stem cell (LSC) persistence remain a clinical concern. Therefore, new therapeutic strategies combining conventional and novel therapies are urgently needed. Since telomerase is involved in oncogenesis and tumor progression but is silent in most human normal somatic cells, it may be an interesting target for CML therapy by selectively targeting cancer cells while minimizing effects on normal cells. Here, we report that hTERT expression is associated with CML disease progression. We also provide evidence that hTERT-deficient K-562 cells do not display telomere shortening and that telomere length is maintained through the ALT pathway. Furthermore, we show that hTERT depletion exerts a growth-inhibitory effect in K- 562 cells and potentiates imatinib through alteration of cell cycle progression leading to a senescence-like phenotype. Finally, we demonstrate that hTERT depletion potentiates the imatinib-induced reduction of the ALDH+-LSC population. Altogether, our results suggest that the combination of telomerase and TKI should be considered as an attractive strategy to treat CML patients to eradicate cancer cells and prevent relapse by tar- geting LSCs

Abstract P3-14-03: The DNA damage repair landscape in Black women with breast cancer

Abstract Black women have 42% higher mortality rate from estrogen receptor positive (ER+) breast cancer than white women. Molecular mechanisms underlying worse outcome in black women are understudied. Recently, downregulation of specific DNA damage repair (DDR) genes was shown to causally induce resistance to standard care endocrine therapy by dysregulating cell cycle regulation, and to associate with poor outcome in white women with ER+ breast cancer. However, frequency and patterns of DDR dysregulation in Black women with ER+ breast cancer and impact on survival outcomes remains untested. By assessing RNA expression of 104 DDR genes across three tumor, and two normal breast, datasets, here, we map for the first time, global patterns of DDR gene expression regulation specific to Black women, both in tumors and in the normal breast. We identify a specific subset of 8 candidate DDR genes that are dysregulated at the RNA level in tumors from Black women. Of note, a novel DDR regulation signature where genes from the homologous recombination pathway are upregulated and genes from SSBR pathways (mainly base excision repair) are coincidently downregulated is almost uniquely detectable in tumors from Black women (8% incidence relative to 1% in tumors from white women, p=0.01). Moreover, this coincident DDR signature associates with dysregulated cell cycle gene expression (p<0.001). In accordance, patients whose tumors demonstrate this coincident DDR signature also have significantly worse survival outcomes across datasets (hazard ratio of 9.5, p<0.001). Overall, these results constitute the first systematic analysis of differences in DDR gene expression regulation between Black and white women and identify a specific DDR signature associated with poor outcome in tumors from Black women. These results provide new grounds for refining biomarker profiles and improving precision medicine for underserved populations. Citation Format: Aloran Mazumder, Athena Jimenez, Rachel E Ellsworth, Sophia George, Stephen J Freedland, Matthew N Bainbridge, Svasti Haricharan. The DNA damage repair landscape in Black women with breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-14-03

Race specific differences in DNA damage repair dysregulation in breast cancer and association with outcome

Abstract IMPORTANCE African American (AA) breast cancer patients have worse outcomes than Caucasian Americans (CAs). DNA damage repair (DDR) genes drive poor outcome in CA estrogen receptor (ER)+ breast cancer patients. Whether DDR genes similarly impact survival in AAs is unknown. Identifying AA-specific patterns of DDR dysregulation could change how we tailor predictive/prognostic biomarkers. OBJECTIVE To characterize DDR dysregulation in ER+ AA patient tumors and test associations with clinical outcome. DESIGN SETTINGS AND PARTICIPANTS Three independent tumor, and two normal breast datasets were analyzed. Tumor datasets: (1) GSE78958 (2) GSE18229 (3) The Cancer Genome Atlas (TCGA). Normal datasets: (4) GSE43973 (5) GSE50939. MAIN OUTCOME AND MEASURES Up/down-regulation of 104 DDR genes was assessed in AA samples vs CAs. Survival associations were assessed for genes dysregulated in multiple datasets. RESULTS Overall, RNA levels of single strand break repair (SSBR) genes were downregulated in AA tumors and double strand break repair (DSBR) genes were upregulated compared to CAs. While SSBR downregulation was mainly detected in tumors, DSBR upregulation was detectable in both tumor and normal breast AA samples. Seven specific DDR genes identified as dysregulated in AAs vs CAs in multiple datasets associated with poor survival. A subset of tumors with simultaneous dysregulation of homologous recombination and single strand break repair genes was enriched in AAs and had associated consistently with poor survival. CONCLUSION AND RELEVANCE Overall, these results constitute the first systematic analysis of differences in DDR regulation in AA ER+ tumors and normal tissue vs CAs. We identify a profile of DDR dysregulation enriched in AA patients, which associates with poor outcome. These results suggest a distinct molecular mechanism of DDR regulation in AAs that lays the groundwork for refining biomarker profiles by race and improving precision medicine for underserved populations. Competing Interest Statement The authors have declared no competing interest. Footnotes * Key Points * Question: Do ER+/HER2− breast tumors in African American women have a distinct pattern of DNA damage repair dysregulation that contribute to poor outcome? * Findings: In this cohort study a distinct DNA damage repair dysregulation was detected in African American women with ER+/HER2− breast cancer. Molecular differences in RNA levels and mutational frequency were observed between African American and Caucasian tumors. Most strikingly, simultaneous downregulation of two DNA repair pathways was enriched in African American patients and associated with poor survival. * Meaning: Disparity in breast cancer outcome between African American and Caucasian women is well established. Our study, for the first time, creates a map of the DNA repair landscape in ER+/HER2− breast cancer from African American women and associates it significantly with worse survival outcome

The DNA damage repair landscape in Black women with breast cancer

Background: Estrogen receptor positive (ER+) breast cancer is one of the most commonly diagnosed malignancies in women irrespective of their race or ethnicity. While Black women with ER+ breast cancer are 42% more likely to die of their disease than White women, molecular mechanisms underlying this disparate outcome are understudied. Recent studies identify DNA damage repair (DDR) genes as a new class of endocrine therapy resistance driver that contributes to poor survival among ER+ breast cancer patients. Here, we systematically analyze DDR regulation in the tumors and normal breast of Black women and its impact on survival outcome. Method: Mutation and up/downregulation of 104 DDR genes in breast tumor and normal samples from Black patients relative to White counterparts was assessed. For DDR genes that were differently regulated in the tumor samples from Black women in multiple datasets associations with survival outcome were tested. Results: Overall, Black patient tumors upregulate or downregulate RNA levels of a wide array of single strand break repair (SSBR) genes relative to their white counterparts and uniformly upregulate double strand break repair (DSBR) genes. This DSBR upregulation was also detectable in samples of normal breast tissue from Black women. Eight candidate DDR genes were reproducibly differently regulated in tumors from Black women and associated with poor survival. A unique DDR signature comprised of simultaneous upregulation of homologous recombination gene expression and downregulation of SSBR genes was enriched in Black patients. This signature associated with cell cycle dysregulation (p < 0.001), a hallmark of endocrine therapy resistance, and concordantly, with significantly worse survival outcomes in all datasets analyzed (hazard ratio of 9.5, p < 0.001). Conclusion: These results constitute the first systematic analysis of DDR regulation in Black women and provide strong rationale for refining biomarker profiles to ensure precision medicine for underserved populations