Parash Parajuli

γ-Tocotrienol-induced autophagy in malignant mammary cancer cells.

γ-Tocotrienol, a member of the vitamin E family of compounds, displays potent antiproliferative and cytotoxic effects in a variety of cancer cell types at treatment doses that have little or no effect on normal cell viability or growth. Autophagy is a tightly regulated lysosomal self-digested process that can either promote cell survival or programmed cell death, but the role of autophagy in mediating γ-tocotrienol-induced cytotoxicity in breast cancer is not presently completely understood. Mouse (+SA) and human (MCF-7 and MDA-MD-231) mammary tumor cells lines were exposed to 0–40 µmol/L γ-tocotrienol for a 24 h treatment period. γ-Tocotrienol treatment caused a relatively large increase in the accumulation of monodansylcadaverine (MDC)-labeled vacuoles, a marker of autophagosome formation, in all tumor cell lines. Results also showed that γ-tocotrienol treatment induced an increased conversion of microtubule-associated protein, 1A/1B-light chain 3, from its cytosolic form (LC3B-I) to its lipidated form (LC3B-II), increased Beclin-1 levels, and increased acridine orange staining as determined by flow cytometry analysis, providing further evidence of γ-tocotrienol-induced autophagy in these mammary cancer cell lines. In contrast, similar treatment with γ-tocotrienol was not found to increase autophagy marker expression in immortalized mouse (CL-S1) and human (MCF-10 A) normal mammary epithelial cell lines. Treatment with γ-tocotrienol also caused a reduction in PI3K/Akt/mTOR signaling and a corresponding increase in the Bax/Bcl-2 ratio, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) levels in these cancer cell lines, suggesting that γ-tocotrienol-induced autophagy may be involved in the initiation of apoptosis. In summary, these findings demonstrate that the cytotoxic effects of γ-tocotrienol are associated with the induction of autophagy in a mouse and human mammary cancer cells.

Potential role of tocotrienols in the treatment and prevention of breast cancer.

Vitamin E is a generic term that refers to a family of compounds that is further divided into two subgroups called tocopherols and tocotrienols. Although all natural forms of vitamin E display potent antioxidant activity, tocotrienols are significantly more potent than tocopherols in inhibiting tumor cell growth and viability, and anticancer activity of tocotrienols is mediated independently of their antioxidant activity. In addition, the anticancer effects of tocotrienols are observed using treatment doses that have little or no effect on normal cell function or viability. This review will summarize experimental studies that have identified the intracellular mechanism mediating the anticancer effects of tocotrienols. Evidence is also provided showing that combined treatment of tocotrienol with other cancer chemotherapies can result in a synergistic inhibition in cancer cell growth and viability. Taken together, these findings strongly indicate that tocotrienols may provide significant health benefits in the prevention and/or treatment of cancer when used either alone as monotherapy or in combination with other anticancer agents.

Anti-proliferative effects of γ-tocotrienol are associated with suppression of c-Myc expression in mammary tumour cells.

Aberrant c‐Myc activity plays a central role in cancer transformation. γ‐tocotrienol is a member of the vitamin E family that displays potent anti‐cancer activity. Here, studies were conducted to determine the role of c‐Myc in mediating anti‐proliferative effects of γ‐tocotrienol in mammary cancer cells.

Optimization of tocotrienols as antiproliferative and antimigratory leads

The vitamin E family members γ- and δ-tocotrienols (2 and 3, respectively) are known natural products with documented anticancer activities. Redox-silent structural modifications, such as esterification, etherification and carbamoylation, of 2 and 3 significantly enhanced their anticancer activities. However, hit-to-lead optimization of tocotrienols and their analogs was yet to be reported at the outset of the project described herein. Subjecting the chroman ring of 2 and 3 to the electrophilic substitution reactions, namely, Mannich and Lederer-Manasse procedures, afforded 42 new products. These included the 3,4-dihydro-1,3-oxazines 3-29 and 35-44, Mannich bases 30-31, and the hydroxymethyl analogs 32-34. Of these, the δ-tocotrienol analogs 8, 11, 18, 24, 25, 27, and 40 inhibited the proliferation of the highly metastatic +SA mammary epithelial cancer cell line, with IC(50) values in the nanomolar (nM) range. In NCIs 60 human tumor cell line panel, 8, 17, 38, and 40 showed antiproliferative activity, with nM GI(50) values. The δ-tocotrienol analogs 10 and 38 inhibited the migration of the highly metastatic human breast cancer cell line MDA-MB-231 with IC(50) values of 1.3 and 1.5 μM, respectively, in the wound-healing assay. A dose of 0.5 mg/day for 14 days of one of the active analogs, 30, significantly slowed the growth of +SA mammary tumors in the syngeneic BALB/c mouse model, compared to the vehicle- and the parent γ-tocotrienol-treated control groups. Electrophilic substitution reactions promoted tocotrienols to lead level and can enable their future use to control metastatic breast malignancies.

Anticancer Effects of γ-Tocotrienol Are Associated with a Suppression in Aerobic Glycolysis

Aerobic glycolysis is an established hallmark of cancer. Neoplastic cells display increased glucose consumption and a corresponding increase in lactate production compared to the normal cells. Aerobic glycolysis is regulated by the phosphatidylinositol-3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) signaling pathway, as well as by oncogenic transcription factors such as c-Myc and hypoxia inducible factor 1α (HIF-1α). γ-Tocotrienol is a natural isoform within the vitamin E family of compounds that displays potent antiproliferative and apoptotic activity against a wide range of cancer cell types at treatment doses that have little or no effect on normal cell viability. Studies were conducted to determine the effects of γ-tocotrienol on aerobic glycolysis in mouse +SA and human MCF-7 breast cancer cells. Treatment with γ-tocotrienol resulted in a dose-responsive inhibition of both +SA and MCF-7 mammary tumor cell growth, and induced a relatively large reduction in glucose utilization, intracellular ATP production and extracellular lactate excretion. These effects were also associated with a large decrease in enzyme expression levels involved in regulating aerobic glycolysis, including hexokinase-II, phosphofructokinase, pyruvate kinase M2, and lactate dehydrogenase A. γ-Tocotrienol treatment was also associated with a corresponding reduction in the levels of phosphorylated (active) Akt, phosphorylated (active) mTOR, and c-Myc, but not HIF-1α or glucose transporter 1 (GLUT-1). In summary, these findings demonstrate that the antiproliferative effects of γ-tocotrienol are mediated, at least in the part, by the concurrent inhibition of Akt/mTOR signaling, c-Myc expression and aerobic glycolysis.

γ-Tocotrienol-induced endoplasmic reticulum stress and autophagy act concurrently to promote breast cancer cell death

The anticancer effects of γ-tocotrienol are associated with the induction of autophagy and endoplasmic reticulum (ER) stress-mediated apoptosis, but a direct relationship between these events has not been established. Treatment with 40 μmol/L of γ-tocotrienol caused a time-dependent decrease in cancer cell viability that corresponds to a concurrent increase in autophagic and endoplasmic reticulum (ER) stress markers in MCF-7 and MDA-MB-231 human breast cancer cells. γ-Tocotrienol treatment was found to cause a time-dependent increase in early phase (Beclin-1, LC3B-II) and late phase (LAMP-1 and cathepsin-D) autophagy markers, and pretreatment with autophagy inhibitors Beclin-1 siRNA, 3-MA or Baf1 blocked these effects. Furthermore, blockage of γ-tocotrienol-induced autophagy with Beclin-1 siRNA, 3-MA, or Baf1 induced a modest, but significant, reduction in γ-tocotrienol-induced cytotoxicity. γ-Tocotrienol treatment was also found to cause a decrease in mitogenic Erk1/2 signaling, an increase in stress-dependent p38 and JNK1/2 signaling, as well as an increase in ER stress apoptotic markers, including phospho-PERK, phospho-eIF2α, Bip, IRE1α, ATF-4, CHOP, and TRB3. In summary, these finding demonstrate that γ-tocotrienol-induced ER stress and autophagy occur concurrently, and together act to promote human breast cancer cell death.

δ-Tocotrienol oxazine derivative antagonizes mammary tumor cell compensatory response to CoCl2-induced hypoxia.

In response to low oxygen supply, cancer cells elevate production of HIF-1α, a hypoxia-inducible transcription factor that subsequently acts to stimulate blood vessel formation and promote survival. Studies were conducted to determine the role of δ-tocotrienol and a semisynthetic δ-tocotrienol oxazine derivative, compound 44, on +SA mammary tumor cell hypoxic response. Treatment with 150 µM CoCl2 induced a hypoxic response in +SA mammary tumor cells as evidenced by a large increase in HIF-1α levels, and combined treatment with compound 44 attenuated this response. CoCl2-induced hypoxia was also associated with a large increase in Akt/mTOR signaling, activation of downstream targets p70S6K and eIF-4E1, and a significant increase in VEGF production, and combined treatment with compound 44 blocked this response. Additional in vivo studies showed that intralesional treatment with compound 44 in BALB/c mice bearing +SA mammary tumors significantly decreased the levels of HIF-1α, and this effect was associated with a corresponding decrease in Akt/mTOR signaling and activation of downstream targets p70S6kinase and eIF-4E1. These findings demonstrate that treatment with the δ-tocotrienol oxazine derivative, compound 44, significantly attenuates +SA mammary tumor cell compensatory responses to hypoxia and suggests that this compound may provide benefit in the treatment of rapidly growing solid breast tumors.

Anticancer Effects of Combined γ-Tocotrienol and PPARγ Antagonist Treatment are Associated with a Suppression in Adipogenic Factor Expression

Cancer cells reprogram their metabolism to meet the demands of accelerated growth. Glucose is the primary source of energy for cancer cells, but under conditions of high-energy demand lipids and free fatty acids become increasingly important. PPAR I³ is a member of the nuclear receptor superfamily and acts to regulate adipocyte differentiation and lipid metabolism. However, in many types of cancer, PPAR I³ activity is elevated in order to increase production of adipogenic factors [1, 2]. I³ -Tocotrienol is an isoform of vitamin E that displays potent anticancer activity [3]. Previous studies have shown that the antiproliferative effects of combined treatment of A£ -tocotrienol with PPAR I³ antagonists was associated with a reduction in PPAR I³ activity, expression of PPAR I³ and RXR, and suppression in Akt activation in MCF-7 and MBA-MB-231 human breast cancer cells [4]. The present study was conducted to determine the effects of combination treatment with these agents on adipogenic factor levels in rapidly proliferating human breast cancer cells. Western blot and qRT-PCR studies showed that combined treatment of I³ -tocotrienol with PPAR I³ antagonists not only suppressed the adipogenic proteins, C/EBPI² and SREBP-1c, but also decreased their target lipogenic enzymes, ap2, FAS, and HMGCoR. However, treatment effects were also observed in PPAR I³ silenced breast cancer cells, indicating that these effects are mediated through PPAR I³ -independent mechanism. These findings suggest the combined treatment of A£ -tocotrienol with PPAR I³ antagonist may have potential as a therapeutic strategy in the treatment of breast cancer.

Abstract P3-03-12: Gamma-tocotrienol induces autophagy in malignant mammary tumor cells

γ-Tocotrienol, a member of the vitamin E family of compounds, displays potent antiproliferative and apoptotic activity in a variety of cancer cell types at treatment doses that have little or no effect on normal cell viability or growth. Autophagy is a tightly regulated lysosomal self-digestive process that can either promote cell survival or be involved in type II cell death. Autophagy is induced in several diseases such as liver disease, neurodegeneration, Crohn9s disease, aging, cancer, and metabolic syndrome. Autophagy is accompanied by the progressive development of vesicle structures from autophagomsomes (not acidic) to amphisome and autolysosomes (acidic). The execution and regulation of the autophagic program relies on several highly conserved autophagy-related genes (the Atg genes) that regulate the cannibalism of intracellular cytoplasm, proteins and organelles. The potential role of autophagy in mediating γ-tocotrienol-induced cytotoxicity is not currently understood. Therefore, studies were conducted to determine the effects of γ-tocotrienol on the induction of autophagy in various breast cancer cell lines. Mouse (+SA) and human (MCF-7 and MDA-MD-231) mammary tumor cells lines were exposed for an acute 24 h period to 20 or 40 μM γ-tocotrienol. Results showed that γ-tocotrienol treatment caused a relatively large increase in the accumulation of monodansylcadaverine (MDC)-labeled vacuoles, an early marker of autophagic compartment formation, in all mammary tumor cell lines. These cells also displayed an increased conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I, the cytosolic form) to LC3-II (lapitated form associated with autophagosome), which is associated with the increased formation of autophagic vacuoles, followed by upregulation of Beclin-1. Treatment with γ-tocotrienol also caused a significant increase in the LC3II/LC3I ratio, a characteristic marker of autophagy, as compared to vehicle-treated control groups. Additional flow cytometery analysis showed that γ-tocotrienol treatment caused an increase in acridine orange staining, an indicator of autophagy, in all mammary cancer cell lines. In contrast, similar treatment of γ-tocotrienol on immortalized mouse (CL-S1) and human (MCF10A) normal mammary epithelial cell lines did not show significant accumulation of MDC–labeled vacuoles or the conversion of LC3-I to LC3-II. In addition, γ-tocotrienol-induced autophagy was also associated with a suppression in PI3K/Akt/mTOR signaling and the initiation of apoptosis as indicated by caspase activation in these mammary cancer cells. In summary, these finding suggest that γ-tocotrienol selectively induces autophagy in mouse and human mammary cancer cell lines and support a novel function of autophagy in promoting breast cancer cells death. This study was supported by grants from First Tech International Ltd., and the Malaysian Palm Oil Council. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-12.

Abstract P3-03-09: Anti-proliferative dose of g-tocotrienol decreases c-Myc stability in breast cancer cells

c-Myc is a helix-loop-helix leucine zipper transcription factor that has essential role in controlling many cell functions, including cell proliferation, differentiation, growth and apoptosis. However, c-Myc overexpression has been reported to occur in a majority of breast cancers and is associated with a poor prognosis. Apart from MYC gene amplification and translocation, it is also tightly regulated by signaling pathway that involves series of interdependent phosphorylation events. Myc stability is increased by phosphorylation at serine 62 (S62) by extracellular signal-regulated kinase (ERK) or cyclin dependent kinase (CDK), whereas subsequent phosphorylation at threonine T58 (T58) by glycogen synthase kinase b (GSK3 β) triggers dephosphorylation of S62 by protein phosphatase 2A-B56α (PP2A- B56α), leading to unbiquitination by SCF-Fbw7 E3 ligase and proteasomal degradation. Additionally, it has been shown that primary human breast cancer cells display increased levels of S62 Myc and decreased level of T58 Myc and mutations in this pathway result in accumulation of high level of oncogenic S62 Myc leading to tumorigenesis. γ-tocotrienol (γT3), a member of the vitamin E family has potent antiproliferative and apoptotic activity in a variety of cancer cell types at treatment doses that have little or no effect on normal cell viability or growth. Additionally, previous studies have shown that anti-proliferative dose of γT3 decreased c-Myc protein level in colorectal and pancreatic cancer cells. Therefore, studies were conducted to determine if γT3 decreases oncogenic S62 phosphorylation and triggers subsequent interdependent phosphorylation leading to c-Myc degradation in neoplastic mouse +SA and MCF-7 human epithelium mammary cancer cell lines. Treatment with 1-8μM γT3 resulted in a dose-responsive inhibition of +SA and MCF-7 breast cancer cell growth. Western blot analysis showed that antiproliferative dose of γ-tocotrienol resulted in a decrease in total c-Myc, phospho S62 Myc and increase in phospho T58 Myc in +SA and MCF-7 breast cancer cells. Further studies showed that similar doses decreased phosphorylated (activated) Akt and its downstream targets GSK-3β and mTOR, as well as phosphorylated (activated) 44/41 MAPK or (Erk 1/2). Additional studies showed that the antiproliferative effects of γT3 were also associated with a decrease in cyclin D1 and cyclin dependent kinase 4 (CDK4). Western blot analysis has also shown an increase in FBw7, an E3 ligase that initiates ubiquitination of c-Myc. However, no change in protein phosphatase 2A (PP2A) and Pin 1 prolyl isomerase was observed in +SA and MCF-7 mammary cancer cells. In summary, these findings demonstrate that the antiproliferative effects of γ-tocotrienol are mediated, at least in part, by decreasing oncogenic c-Myc (S62) levels and a corresponding reduction in Akt/mTOR and MAPK signaling. These effects were also associated with an increase in GSK-3β-induced phosphorylation of T58 and the promotion of the ubiquitination and degradation of c-Myc. This study was supported by grants from First Tech International Ltd., and the Malaysian Palm Oil Council. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-09.