Jasdeep K. Saggar

Flaxseed and Pure Secoisolariciresinol Diglucoside, but Not Flaxseed Hull, Reduce Human Breast Tumor Growth (MCF-7) in Athymic Mice

Previous studies have shown that dietary flaxseed (FS) can reduce the growth of established human breast tumors in athymic mice with low circulating estrogen concentrations. In this study, we determined the effect of FS compared with pure lignan at the level it is present in FS [secoisolariciresinol diglucoside (SDG)] and to the lignan-rich fraction [FS hull (FH)] on human breast tumor growth and their potential mechanisms of action. Ovariectomized, athymic mice, each with an implanted 17 beta-estradiol (E2) pellet (0.36 mg), were injected with human estrogen receptor (ER) positive breast cancer cells (MCF-7). When tumors were established, the E2 pellet was removed. Mice were fed either the control basal diet (BD), FS (100 g/kg diet), SDG (1 g/kg diet), or FH (18 g/kg diet) for 8 wk. Compared with the BD, FS and SDG significantly decreased the palpable tumor size, but effects of FS, SDG, and FH did not differ from one another. All treatments significantly inhibited cell proliferation, but only FS and SDG induced significantly higher apoptosis. Both FS and SDG significantly decreased mRNA expressions of Bcl2, cyclin D1, pS2, ERalpha, and ERbeta, epidermal growth factor receptor, and insulin-like growth factor receptor. FS also reduced human epidermal growth factor receptor 2 mRNA and SDG decreased phospho-specific mitogen-activated protein kinase expression. FH did not significantly reduce these biomarkers. In conclusion, pure SDG has a similar effect as FS in reducing tumor growth and in mechanisms of action, including downregulating ER- and growth factor-mediated cell signaling. The lesser effects of FH indicate a need for a higher dose to be more effective.

The Effect of Secoisolariciresinol Diglucoside and Flaxseed Oil, Alone and in Combination, on MCF-7 Tumor Growth and Signaling Pathways

Flaxseed (FS), an oilseed containing high amounts of the phytoestrogen lignan, secoisolariciresinol diglucoside (SDG), and n-3 fatty acid, α-linolenic acid-rich oil (FO), has been shown to inhibit the growth of established human breast tumors (MCF-7) in ovariectomized (OVX) athymic mice. However, the major FS component responsible for this effect and the mechanism(s) of its action are unclear. Hence, this study determined, in a 2 × 2 factorial design, the effect of SDG and FO, alone or in combination, on the growth of established human estrogen receptor positive (ER+) breast tumors and the potential mechanism(s) of its action. OVX mice with established ER+ human breast tumors (MCF-7) were treated for 8 wk with basal diet (BD, control) or BD supplemented with SDG (1 g/kg), FO (38.5 g/kg), or SDG + FO. All treatments reduced the tumor growth, but SDG had the greatest effect primarily through reducing tumor cell proliferation rather than increasing apoptosis. SDG had a main effect in the reduction of PS2, BCL2, and IGF-1R mRNA expression, whereas FO had a main effect only in PAKT reduction. SDG alone also lowered the ERα, ERβ, EGFR, BCL2 mRNA, and PMAPK protein, indicating that its effect involves the modulation of the ER– and growth factor receptor-mediated signaling pathways.

Dietary flaxseed lignan or oil combined with tamoxifen treatment affects MCF-7 tumor growth through estrogen receptor- and growth factor-signaling pathways.

This study aimed to elucidate which component of flaxseed, i.e. secoisolariciresinol diglucoside (SDG) lignan or flaxseed oil (FO), makes tamoxifen (TAM) more effective in reducing growth of established estrogen receptor positive breast tumors (MCF-7) at low circulating estrogen levels, and potential mechanisms of action. In a 2 x 2 factorial design, ovariectomized athymic mice with established tumors were treated for 8 wk with TAM together with basal diet (control), or basal diet supplemented with SDG (1 g/kg diet), FO (38.5 g/kg diet), or combined SDG and FO. SDG and FO were at levels in 10% flaxseed diet. Palpable tumors were monitored and after animal sacrifice, analyzed for cell proliferation, apoptosis, ER-mediated (ER-alpha, ER-beta, trefoil factor 1, cyclin D1, progesterone receptor, AIBI), growth factor-mediated (epidermal growth factor receptor, human epidermal growth factor receptor-2, insulin-like growth factor receptor-1, phosphorylated mitogen activated protein kinase, PAKT, BCL2) signaling pathways and angiogenesis (vascular endothelial growth factor). All treatments reduced the growth of TAM-treated tumors by reducing cell proliferation, expression of genes, and proteins involved in the ER- and growth factor-mediated signaling pathways with FO having the greatest effect in increasing apoptosis compared with TAM treatment alone. SDG and FO reduced the growth of TAM-treated tumors but FO was more effective. The mechanisms involve both the ER- and growth factor-signaling pathways.

Activity of the hypoxia-activated pro-drug TH-302 in hypoxic and perivascular regions of solid tumors and its potential to enhance therapeutic effects of chemotherapy

Many chemotherapy drugs have poor therapeutic activity in regions distant from tumor blood vessels because of poor tissue penetration and low cytotoxic activity against slowly‐proliferating cells. The hypoxia‐activated pro‐drug TH‐302 may have selective toxicity for hypoxic and neighboring cells in tumors. Here we characterize the spatial distribution and ability of TH‐302 to selectively target hypoxic regions and complement the effect of doxorubicin and docetaxel by modifying biomarker distribution. Athymic nude mice bearing human breast MCF‐7 or prostate PC‐3 tumors were treated with doxorubicin or docetaxel respectively and TH‐302 alone or in combination. Biomarkers of drug effect including γH2aX (a marker of DNA damage), cleaved caspase‐3 or ‐6 (markers of apoptosis) and reduction in Ki‐67 (a marker of cell proliferation) were quantified in tumor sections in relation to functional blood vessels (recognized by DiOC7) and hypoxia (recognized by EF5) using immunohistochemistry. γH2aX expression at 10 min and cleaved caspase‐3 or ‐6 at 24 hr after doxorubicin or docetaxel decreased with increasing distance from tumor blood vessels, with minimal expression in hypoxic regions; maximum reduction in Ki67 levels was observed in regions closest to vasculature at 24 hr. TH‐302 induced maximal cell damage in hypoxic and neighboring regions, but was also active in tumor regions closer to blood vessels. TH‐302 given 4 hr before doxorubicin or docetaxel increased DNA damage and apoptosis throughout the tumor compared to chemotherapy alone. When given with doxorubicin or docetaxel, TH‐302 complements and enhances anticancer effects in both perivascular and hypoxic regions but also increases toxicity.

Chemotherapy Rescues Hypoxic Tumor Cells and Induces Their Reoxygenation and Repopulation—An Effect That Is Inhibited by the Hypoxia-Activated Prodrug TH-302

Purpose: Chemotherapy targets rapidly proliferating tumor cells, but spares slowly proliferating hypoxic cells. We hypothesized that nutrition of hypoxic cells would improve in intervals between chemotherapy, and that hypoxic cells destined to die without treatment would survive and proliferate. Experimental Design: We therefore evaluated repopulation and reoxygenation following chemotherapy, and the effects of the hypoxia-activated prodrug TH-302 on these processes. Tumor-bearing mice were treated with doxorubicin or docetaxel ± TH-302. Pimonidazole (given concurrent with chemotherapy) and EF5 (given 24 to 120 hours later) identified hypoxic cells. Proliferation (Ki67) and oxygen status (EF5 uptake) of formerly hypoxic (pimo positive) cells were quantified by immunohistochemistry. Results: Chronically hypoxic cells had limited proliferation in control tumors. After chemotherapy, we observed reoxygenation and increased proliferation of previously hypoxic cells; these processes were inhibited by TH-302. Conclusions: Chemotherapy leads to paradoxical sparing of hypoxic cells destined to die in solid tumors in absence of treatment, and their reoxygenation and proliferation: TH-302 inhibits these processes. Clin Cancer Res; 21(9); 2107–14. ©2015 AACR.

Flaxseed cotyledon fraction reduces tumour growth and sensitises tamoxifen treatment of human breast cancer xenograft (MCF-7) in athymic mice

Dietary flaxseed (FS) inhibited the growth of human breast tumours and enhanced the effectiveness of tamoxifen (TAM) in athymic mice with low oestradiol (E2) levels. The present study determined whether the n-3 fatty acid-rich cotyledon fraction of FS (FC), alone or in combination with TAM, has a similar effect and thus can substitute for FS. In a 2 × 2 factorial design, ovariectomised mice with established oestrogen receptor (ER)-positive breast tumours (MCF-7) were treated as follows: groups 1 and 2 were fed the basal diet (BD, control) and FC diet (82 g FC/kg), respectively. Groups 3 and 4 with TAM implants (5 mg) were fed the BD and FC diet, respectively. At 8 weeks post-treatment, mice were euthanised, and tumours were analysed by immunohistochemistry and real-time PCR. BD, FC and FC/TAM groups significantly decreased tumour area, but the TAM group did not. Tumour regression in the FC/TAM group was greater compared to the TAM group. FC lowered cell proliferation but had no effect on apoptosis; the opposite was observed with TAM. FC suppressed mRNA expressions of pS2 and insulin-like growth factor 1 receptor (IGF-1R) and protein expressions of ERα, phosphospecific ERα, human epidermal growth factor receptor 2 (HER2), phosphospecific HER2 (pHER2) and amplified in breast 1 (AIB1), while TAM up-regulated mRNA expressions of Bcl2, progesterone receptor and IGF-1R and protein expression of pHER2, and down-regulated ERβ mRNA. FC modulated the effect of TAM on tumour growth biomarkers. In conclusion, FC reduced the growth of ER+ human breast tumours at low circulating E2, alone and combined with TAM, in part through modulation of ER- and growth factor-mediated signalling pathways; it may substitute for FS in increasing the effectiveness of TAM.

Mechanisms of Drug Resistance Related to the Microenvironment of Solid Tumors and Possible Strategies to Inhibit Them.

AbstractDrug resistance can occur at the individual cellular level or as a result of properties of the tumor microenvironment. The convoluted vasculature within tumors results in robustly proliferating well-nourished cells located proximal to functional blood vessels and regions of slowly proliferating (often hypoxic) cells located distal to functional blood vessels. Irregular blood flow and large distances between functional blood vessels in solid tumors lead to poor drug distribution within them such that cells distal from functional blood vessels are exposed to ineffective concentrations of drug, resulting in therapeutic resistance. Strategies to improve or complement the distribution of anticancer drugs within tumors hold promise for increasing antitumor effects without corresponding increases in normal tissue toxicity. In particular, use of hypoxia-targeted agents and modulation of autophagy have shown promising results in enhancing the distribution of drug activity within solid tumors and hence antitumor efficacy. In this review, we describe causes of resistance to chemotherapy that relate to the microenvironment of solid tumors and the potential to improve antitumor effects by countering such mechanisms of resistance.

Use of Molecular Biomarkers to Quantify the Spatial Distribution of Effects of Anticancer Drugs in Solid Tumors

Poor distribution of anticancer drugs within solid tumors may limit their effectiveness. Here, we characterize the distribution within solid tumors of biomarkers of drug effect. γ-H2AX, cleaved-caspase-3 or -6, and Ki67 were quantified in tumor sections in relation to blood vessels (recognized by CD31) using monoclonal antibodies and immunohistochemistry. To validate their use, we compared their time-dependent distribution with that of (i) fluorescent doxorubicin and (ii) a monoclonal antibody that detects melphalan-induced DNA adducts. The biomarkers were then used to quantify the distribution of docetaxel in relation to tumor blood vessels. Activation of γ-H2AX was evaluated following in vitro exposure of tumor cells to multiple drugs. Distributions of doxorubicin in MDA-MB-231 and MCF-7 xenografts and of melphalan-induced DNA adducts in MCF-7 and EMT-6 tumors decreased with distance from blood vessels, similar to the distributions of (i) γ-H2AX at 10 minutes, (ii) cleaved caspase-3 or -6, and (iii) change in Ki67 at 24 hours following treatment. The distribution of these biomarkers following treatment with docetaxel also decreased with increasing distance from tumor blood vessels. Activation of γ-H2AX occurred within 1 hour after exposure to several drugs in culture. Multiple anticancer drugs show a decrease in activity with increasing distance from tumor blood vessels; poor drug distribution is an important cause of drug resistance. The above biomarkers may be used in designing strategies to overcome therapeutic resistance by modifying or complementing the limited spatial distribution of drug activity in solid tumors. Mol Cancer Ther; 12(4); 542–52. ©2013 AACR.

Effects of flaxseed lignan and oil on bone health of breast-tumor-bearing mice treated with or without tamoxifen.

Previous studies showed that flaxseed lignan (secoisolariciresinol diglucoside, SDG) and oil (FO) inhibit established breast tumor growth in athymic mice with or without tamoxifen (TAM) treatment. TAM was found to increase bone mineral content (BMC) and density (BMD) in breast cancer patients. It is not known whether SDG or FO alone or combined with TAM affects bone health. Hence, the effects of SDG and FO, alone or in combination, on BMC, BMD, and biomechanical bone strength in ovariectomized athymic mice with established human breast tumors (MCF-7) treated with or without TAM were studied. In a factorial design, mice were divided into four non-TAM and four TAM groups. Each group consisted of mice fed a basal diet (BD), SDG (1 g/kg), FO (38.5 g/kg) or SDG + FO (combination) diets. The TAM group had TAM implants that provide a 5-mg TAM dose released over 60 d. TAM exerted an overall significant effect in increasing BMC, BMD, and biomechanical strength in femurs and lumbar vertebra. Without TAM treatment, SDG produced significant lower femur BMD (6%) while FO produced lower vertebrae BMC (8%) and BMD (6%). With TAM treatment, SDG and FO did not exert an effect on BMC and BMD at the femur or vertebra. SDG and FO produced no marked effect on biomechanical bone strength with or without TAM treatment. In conclusion, FS components did not significantly attenuate the positive effects on bone induced by TAM in this model system, indicating no apparent adverse effects on bone health.

Abstract A54: Pantoprazole enhances the activity of docetaxel chemotherapy for solid tumors by inhibition of autophagy

Purpose: Resistance to anti-tumour agents is a major cause of treatment failure in patients with cancer. Luciani and colleagues (JNCI 2004; 96:1702-13) have shown that PPIs can augment the effects of chemotherapy in vitro and in vivo, most likely by modifying the acid-base balance across the cell membrane and/or of intracellular organelles such as lysosomes and endososmes. Endosomes are important intermediaries of the process of autophagy, a survival mechanism for stressed cells and a possible target for anticancer therapy. Here we evaluated the effect of pantoprazole (PTP) (i) to modify cytotoxic effects of the commonly-used anticancer drug docetaxel (DOC) for cultured cells and for human tumour xenografts. (ii) to modify the distribution of biomarkers of activity of docetaxel in relation to tumour blood vessels. (iii) to inhibit autophagy. Methods: Endosomal pH of cells was measured by fluorescence spectroscopy following exposure to the endosomal probe Lysosensor. Levels of autophagy were evaluated by quantifying the level of the LC3B protein in Western blots. Human prostate cancer PC3 cells were transfected with a construct where LC3B was linked to red (RFP) and green (GFP) fluorescent protein (mRFP-GFP-LC3) such that cells with high levels of autophagy stained red while those with low levels stained green. Cytotoxicity of DOC +/-PTP was evaluated in vitro by a colony-forming assay for PC3 parental cells, and for cells transfected with shRNAs to inhibit autophagy. Growth delay of three human xenografts in nude mice was evaluated following treatment with single or multiple doses of DOC+/- PTP 3. The distribution of drug effects in tumour sections in relation to functional blood vessels (recognized by Dioc7) was quantified (at early intervals of ~10 minutes) by γH2AX, a marker of DNA damage, (at 24 hours) by cleaved caspase 3, a marker of apoptosis, Ki67, a marker of cell proliferation and LC3, a marker of autophagy. Results: PTP increased endosomal pH and inhibited autophagy in a dose and time dependent manner. PTP was not cytotoxic when used alone but increased the cytotoxicity of DOC for cultured PC3 cells; the effects of PTP were reduced against genetically-modified cells that have reduced ability to undergo autophagy. Pre-treatment with PTP increased markedly the growth delay due to DOC of three different xengografts, especially after three treatments at weekly intervals, with minimal effects on DOC toxicity. Cellular effects of DOC evaluated by γH2AX and by cleaved caspase 3 decreased with increasing distance from tumour blood vessels, but were enhanced substantially by pre-treatment with PTP. There is less cell proliferation and marked reduction in hypoxia in xenografts generated from autophagy-deficient PC3 cells compared to PC3 wild type xenografts. Conclusions: Autophagy is a mechanism of resistance of chemotherapy that may be modified by pantoprazole to improve therapeutic. Docetaxel is the primary chemotherapy for men with metastatic castration resistant prostate cancer, and we plan a phase II study of PTP + DOC in such patients. Citation Format: QIAN TAN, Anthony Joshua, Jasdeep K. Saggar, Yu Man, Carol CM Lee, Bradly R. Wouters, Ian F. Tannock. Pantoprazole enhances the activity of docetaxel chemotherapy for solid tumors by inhibition of autophagy. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A54. doi:10.1158/1538-7445.CHTME14-A54