The application of autologous cancer immunotherapies in the age of memory-NK cells.

Cellular immunotherapy has revolutionized the oncology field, yielding improved results against hematological and solid malignancies. NK cells have become an attractive alternative due to their capacity to activate upon recognition of "stress" or "danger" signals independently of Major Histocompatibility Complex (MHC) engagement, thus making tumor cells a perfect target for NK cell-mediated cancer immunotherapy even as an allogeneic solution. While this allogeneic use is currently favored, the existence of a characterized memory function for NK cells ("memory-like" NK cells) advocates for an autologous approach, that would benefit from the allogeneic setting discoveries, but with added persistence and specificity. Still, both approaches struggle to exert a sustained and high anticancer effect in-vivo due to the immunosuppressive tumor micro-environment and the logistical challenges of cGMP production or clinical deployment. Novel approaches focused on the quality enhancement and the consistent large-scale production of highly activated therapeutic memory-like NK cells have yielded encouraging but still unconclusive results. This review provides an overview of NK biology as it relates to cancer immunotherapy and the challenge presented by solid tumors for therapeutic NKs. After contrasting the autologous and allogeneic NK approaches for solid cancer immunotherapy, this work will present the current scientific focus for the production of highly persistent and cytotoxic memory-like NK cells as well as the current issues with production methods as they apply to stress-sensitive immune cells. In conclusion, autologous NK cells for cancer immunotherapy appears to be a prime alternative for front line therapeutics but to be successful, it will be critical to establish comprehensives infrastructures allowing the production of extremely potent NK cells while constraining costs of production.

Metabolism of [6]-shogaol in mice and in cancer cells.

Ginger has received extensive attention because of its antioxidant, anti-inflammatory, and antitumor activities. However, the metabolic fate of its major components is still unclear. In the present study, the metabolism of [6]-shogaol, one of the major active components in ginger, was examined for the first time in mice and in cancer cells. Thirteen metabolites were detected and identified, seven of which were purified from fecal samples collected from [6]-shogaol-treated mice. Their structures were elucidated as 1-(4'-hydroxy-3'-methoxyphenyl)-4-decen-3-ol (M6), 5-methoxy-1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M7), 3',4'-dihydroxyphenyl-decan-3-one (M8), 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-ol (M9), 5-methylthio-1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M10), 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M11), and 5-methylthio-1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-ol (M12) on the basis of detailed analysis of their (1)H, (13)C, and two-dimensional NMR data. The rest of the metabolites were identified as 5-cysteinyl-M6 (M1), 5-cysteinyl-[6]-shogaol (M2), 5-cysteinylglycinyl-M6 (M3), 5-N-acetylcysteinyl-M6 (M4), 5-N-acetylcysteinyl-[6]-shogaol (M5), and 5-glutathiol-[6]-shogaol (M13) by analysis of the MS(n) (n = 1-3) spectra and comparison to authentic standards. Among the metabolites, M1 through M5, M10, M12, and M13 were identified as the thiol conjugates of [6]-shogaol and its metabolite M6. M9 and M11 were identified as the major metabolites in four different cancer cell lines (HCT-116, HT-29, H-1299, and CL-13), and M13 was detected as a major metabolite in HCT-116 human colon cancer cells. We further showed that M9 and M11 are bioactive compounds that can inhibit cancer cell growth and induce apoptosis in human cancer cells. Our results suggest that 1) [6]-shogaol is extensively metabolized in these two models, 2) its metabolites are bioactive compounds, and 3) the mercapturic acid pathway is one of the major biotransformation pathways of [6]-shogaol.

Inhibition of human breast cancer xenograft growth by cruciferous vegetable constituent benzyl isothiocyanate.

Benzyl isothiocyanate (BITC), a constituent of cruciferous vegetables such as garden cress, inhibits growth of human breast cancer cell lines in culture. The present study was undertaken to determine in vivo efficacy of BITC against MDA-MB-231 human breast cancer xenografts. The BITC administration retarded growth of MDA-MB-231 cells subcutaneously implanted in female nude mice without causing weight loss or any other side effects. The BITC-mediated suppression of MDA-MB-231 xenograft growth correlated with reduced cell proliferation as revealed by immunohistochemical analysis for Ki-67 expression. Analysis of the vasculature in the tumors from BITC-treated mice indicated smaller vessel area compared with control tumors based on immunohistochemistry for angiogenesis marker CD31. The BITC-mediated inhibition of angiogenesis in vivo correlated with downregulation of vascular endothelial growth factor (VEGF) receptor 2 protein levels in the tumor. Consistent with these results, BITC treatment suppressed VEGF secretion and VEGF receptor 2 protein levels in cultured MDA-MB-231 cells. Moreover, the BITC-treated MDA-MB-231 cells exhibited reduced capacity for migration compared with vehicle-treated control cells. In contrast to cellular data, BITC administration failed to elicit apoptotic response as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. In conclusion, the present study demonstrates in vivo anti-cancer efficacy of BITC against MDA-MB-231 xenografts in association with reduced cell proliferation and suppression of neovascularization. These preclinical observations merit clinical investigation to determine efficacy of BITC against human breast cancers.

Erythropoietin signaling promotes transplanted progenitor cell survival.

We examine the potential for erythropoietin signaling to promote donor cell survival in a model of myoblast transplantation. Expression of a truncated erythropoietin receptor in hematopoietic stem cells has been shown to promote selective engraftment in mice. We previously demonstrated expression of endogenous erythropoietin receptor on murine myoblasts, and erythropoietin treatment can stimulate myoblast proliferation and delay differentiation. Here, we report that enhanced erythropoietin receptor expression, as well as exogenous erythropoietin treatment in myoblasts, provided a survival advantage and protection against apoptosis under serum-starvation conditions. When cultured in differentiation medium, expression of the myogenic regulatory proteins shifted toward early differentiation with increased erythropoietin receptor. Expression of early myogenic differentiation proteins Myf-5 and MyoD increased, while later stage protein myogenin decreased. Transplantation of C2C12 myoblasts overexpressing truncated erythropoietin receptor showed more transplanted cell incorporation into muscle fibers in muscular dystrophy mdx mice. These cells also restored dystrophin protein expression in mdx mice at 6 wk after cell treatment that was further increased with exogenous erythropoietin administration. In summary, enhanced erythropoietin receptor expression promotes transplanted cell survival in a mouse model for myoblast transplantation and provides dystrophin expression in mice with muscular dystrophy.

The human beta-globin locus control region can silence as well as activate gene expression.

Using recombinase-mediated cassette exchange to test multiple transgenes at the same site of integration, we demonstrate a novel chromatin context-dependent silencer activity of the beta-globin locus control region (LCR). This silencer activity requires DNase I hypersensitive sites HS2 and HS3 but not HS4. After silencing, the silenced cassettes adopt a typical closed chromatin conformation (histone H3 and H4 deacetylation, histone H3-K4 methylation, DNA methylation, and replication in late S phase). In the absence of the LCR at the same site of integration, the chromatin remains decondensed. We demonstrate that the LCR is necessary but not sufficient to trigger these chromatin changes. We also provide evidence that this novel silencing activity is caused by transcriptional interference triggered by activation of transcription in the flanking sequences by the LCR.

Carbohydrate Intake Does Not Counter the Post-Exercise Decrease in Natural Killer Cell Cytotoxicity.

In a study using a randomized crossover approach, cyclists (n = 20, overnight fasted) engaged in three 75 km time trials while ingesting water (WAT) or carbohydrate (0.2 g/kg every 15 min) from bananas (BAN) or a 6% sugar beverage (SUG). Blood samples were collected pre-exercise and 0 h, 1.5 h, and 21 h post-exercise and analyzed for natural killer (NK) cytotoxicity activity (NKCA) using pure NK cell populations. The two carbohydrate trials (BAN, SUG) compared to WAT were associated with higher post-exercise glucose and lower cortisol, total blood leukocyte, neutrophil, and NK cell counts (interaction effects, p < 0.001). The immediate post-exercise increase in NK cell counts was higher in WAT (78%) compared to BAN (32%) and SUG (15%) trials (p ≤ 0.017). The 1.5 h post-exercise decrease in NK cell counts did not differ after WAT (-46%), BAN (-46%), and SUG (-51%) trials. The pattern of change in post-exercise NKCA differed between trials (p < 0.001). The 1.5 h post-exercise decreases in NKCA were 23%, 29%, and 33% in the WAT, BAN, and SUG trials, respectively, but trial contrasts did not differ significantly. Carbohydrate ingestion from BAN or SUG attenuated immediate post-exercise increases in leukocyte, neutrophil, and NK cell counts, but did not counter the 1.5 h decreases in NK cell counts and NKCA.

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user's errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3'-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data's integrity.

Cysteine-conjugated metabolites of ginger components, shogaols, induce apoptosis through oxidative stress-mediated p53 pathway in human colon cancer cells.

Shogaols, the major constituents of thermally processed ginger, have been proven to be highly effective anticancer agents. Our group has identified cysteine-conjugated shogaols (M2, M2', and M2″) as the major metabolites of [6]-, [8]-, and [10]-shogaol in human and found that M2 is a carrier of its parent molecule [6]-shogaol in cancer cells and in mice, while being less toxic to normal colon fibroblast cells. The objectives of this study are to determine whether M2' and M2″ behave in a similar manner to M2, in both metabolism and efficacy as anticancer agents, and to further explore the biological pro-apoptotic mechanisms of the cysteine-conjugated shogaols against human colon cancer cells HCT-116 and HT-29. Our results show that [8]- and [10]-shogaol have similar metabolic profiles to [6]-shogaol and exhibit similar toxicity toward human colon cancer cells. M2' and M2″ both show low toxicity against normal colon cells but retain potency against colon cancer cells, suggesting that they have similar activity to M2. We further demonstrate that the cysteine-conjugated shogaols can cause cancer cell death through the activation of the mitochondrial apoptotic pathway. Our results show that oxidative stress activates a p53 pathway that ultimately leads to p53 up-regulated modulator of apoptosis (PUMA) induction and down-regulation of B-cell lymphoma 2 (Bcl-2), followed by cytochrome c release, perturbation of inhibitory interactions of X-linked inhibitor of apoptosis protein (XIAP) with caspases, and finally caspase 9 and 3 activation and cleavage. A brief screen of the markers attenuated by the proapoptotic activity of M2 revealed similar results for [8]- and [10]-shogaol and their respective cysteine-conjugated metabolites M2' and M2″. This study highlights the cysteine-conjugated metabolites of shogaols as novel dietary colon cancer preventive agents.

Induction of lung cancer cell apoptosis through a p53 pathway by [6]-shogaol and its cysteine-conjugated metabolite M2.

Dietary chemoprevention of cancer offers the possibility to suppress or inhibit cancer growth before it develops into more advanced and lethal stages. To this end, identification of novel compounds and their mechanisms of action is constantly needed. In this study, we describe that a major component of dry ginger (Zingiber officinalis), [6]-shogaol (6S), can be quickly metabolized in A549 human lung cancer cell line. One of the resulting metabolites, the cysteine-conjugated 6S (M2), exhibits toxicity to cancer cells similar to the parent compound 6S, but is relatively less toxic toward normal cells than 6S. We further demonstrate that both compounds can cause cancer cell death by activating the mitochondrial apoptotic pathway. Our results show that the cancer cell toxicity is initiated by early modulation of glutathione (GSH) intracellular content. The subsequently generated oxidative stress activates a p53 pathway that ultimately leads to the release of mitochondria-associated apoptotic molecules such as cytochrome C, and cleaved caspases 3 and 9. In a xenograft nude mouse model, a dose of 30 mg/kg of 6S or M2 was able to significantly decrease tumor burden, without any associated toxicity to the animals. This effect was correlated with an induction of apoptosis and reduction of cell proliferation in the tumor tissues. Taken together, our results show that 6S metabolism is an integral part of its anticancer activities in vitro and in vivo. This allows us to characterize M2 as a novel compound with superior in vivo chemopreventive properties that targets similar anticancer mechanisms as 6S.

Metabolites of ginger component [6]-shogaol remain bioactive in cancer cells and have low toxicity in normal cells: chemical synthesis and biological evaluation.

Our previous study found that [6]-shogaol, a major bioactive component in ginger, is extensively metabolized in cancer cells and in mice. It is unclear whether these metabolites retain bioactivity. The aim of the current study is to synthesize the major metabolites of [6]-shogaol and evaluate their inhibition of growth and induction of apoptosis in human cancer cells. Twelve metabolites of [6]-shogaol (M1, M2, and M4-M13) were successfully synthesized using simple and easily accessible chemical methods. Growth inhibition assays showed that most metabolites of [6]-shogaol had measurable activities against human cancer cells HCT-116 and H-1299. In particular, metabolite M2 greatly retained the biological activities of [6]-shogaol, with an IC(50) of 24.43 µM in HCT-116 human colon cancer cells and an IC(50) of 25.82 µM in H-1299 human lung cancer cells. Also exhibiting a relatively high potency was thiol-conjugate M13, with IC(50) values of 45.47 and 47.77 µM toward HCT-116 and H-1299 cells, respectively. The toxicity evaluation of the synthetic metabolites (M1, M2, and M4-M13) against human normal fibroblast colon cells CCD-18Co and human normal lung cells IMR-90 demonstrated a detoxifying metabolic biotransformation of [6]-shogaol. The most active metabolite M2 had almost no toxicity to CCD-18Co and IMR-90 normal cells with IC(50)s of 99.18 and 98.30 µM, respectively. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay indicated that apoptosis was triggered by metabolites M2, M13, and its two diastereomers M13-1 and M13-2. There was no significant difference between the apoptotic effect of [6]-shogaol and the effect of M2 and M13 after 6 hour treatment.

Prevention of mammary carcinogenesis in MMTV-neu mice by cruciferous vegetable constituent benzyl isothiocyanate.

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of human breast cancer cells in culture. The present study provides in vivo evidence for efficacy of BITC for prevention of mammary cancer in MMTV-neu mice. Administration of BITC at 1 and 3 mmol/kg diet for 25 weeks markedly suppressed the incidence and/or burden of mammary hyperplasia and carcinoma in female MMTV-neu mice without causing weight loss or affecting neu protein level. For example, cumulative incidence of hyperplasia/carcinoma was significantly lower in mice fed BITC-supplemented diets compared with control mice (P = 0.01 by Fisher's test). The BITC-mediated prevention of mammary carcinogenesis correlated with suppression of cell proliferation and increased apoptosis. The average number of Ki-67-positive cells in the carcinoma lesions of 3 mmol BITC group was lower by approximately 21% (P < 0.05) compared with tumors from control mice. Apoptotic bodies in the mammary tumor were higher by about 2- to 2.5-fold in the 1 and 3 mmol BITC treatment groups (P < 0.05) compared with control group. The BITC administration also resulted in overexpression of E-cadherin and infiltration of CD3(+) T-cells in the tumor. Although BITC treatment increased cytotoxicity of natural killer (NK) cells in vitro, dietary feeding of BITC failed to augment NK cell lytic activity in an ex vivo assay. The present study demonstrating efficacy of BITC against mammary cancer in an animal model provides impetus to determine its activity in a clinical setting.

Sulforaphane inhibits prostate carcinogenesis and pulmonary metastasis in TRAMP mice in association with increased cytotoxicity of natural killer cells.

The present study shows that oral gavage of 6 mumol d,l-sulforaphane (SFN), a synthetic analogue of cruciferous vegetable-derived L isomer, thrice per week beginning at 6 weeks of age, significantly inhibits prostate carcinogenesis and pulmonary metastasis in TRAMP mice without causing any side effects. The incidence of the prostatic intraepithelial neoplasia and well-differentiated (WD) carcinoma were approximately 23% to 28% lower (P < 0.05 compared with control by Mann-Whitney test) in the dorsolateral prostate (DLP) of SFN-treated mice compared with controls, which was not due to the suppression of T-antigen expression. The area occupied by the WD carcinoma was also approximately 44% lower in the DLP of SFN-treated mice relative to that of control mice (P = 0.0011 by Mann Whitney test). Strikingly, the SFN-treated mice exhibited approximately 50% and 63% decrease, respectively, in pulmonary metastasis incidence and multiplicity compared with control mice (P < 0.05 by t test). The DLP from SFN-treated mice showed decreased cellular proliferation and increased apoptosis when compared with that from control mice. Additionally, SFN administration enhanced cytotoxicity of cocultures of natural killer (NK) cells and dendritic cells (DC) against TRAMP-C1 target cells, which correlated with infiltration of T cells in the neoplastic lesions and increased levels of interleukin-12 production by the DC. In conclusion, the results of the present study indicate that SFN administration inhibits prostate cancer progression and pulmonary metastasis in TRAMP mice by reducing cell proliferation and augmenting NK cell lytic activity.

Withaferin A causes FOXO3a- and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo.

Withaferin A (WA) is derived from the medicinal plant Withania somnifera, which has been safely used for centuries in Indian Ayurvedic medicine for treatment of different ailments. We now show, for the first time, that WA exhibits significant activity against human breast cancer cells in culture and in vivo. The WA treatment decreased viability of MCF-7 (estrogen-responsive) and MDA-MB-231 (estrogen-independent) human breast cancer cells in a concentration-dependent manner. The WA-mediated suppression of breast cancer cell viability correlated with apoptosis induction characterized by DNA condensation, cytoplasmic histone-associated DNA fragmentation, and cleavage of poly-(ADP-ribose)-polymerase. On the other hand, a spontaneously immortalized normal mammary epithelial cell line (MCF-10A) was relatively more resistant to WA-induced apoptosis compared with breast cancer cells. The WA-mediated apoptosis was accompanied by induction of Bim-s and Bim-L in MCF-7 cells and induction of Bim-s and Bim-EL isoforms in MDA-MB-231 cells. The cytoplasmic histone-associated DNA fragmentation resulting from WA exposure was significantly attenuated by knockdown of protein levels of Bim and its transcriptional regulator FOXO3a in both cell lines. Moreover, FOXO3a knockdown conferred marked protection against WA-mediated induction of Bim-s expression. The growth of MDA-MB-231 cells implanted in female nude mice was significantly retarded by 5 weekly i.p. injections of 4 mg WA/kg body weight. The tumors from WA-treated mice exhibited reduced cell proliferation and increased apoptosis compared with tumors from control mice. These results point toward an important role of FOXO3a and Bim in regulation of WA-mediated apoptosis in human breast cancer cells.

Garlic constituent diallyl trisulfide prevents development of poorly differentiated prostate cancer and pulmonary metastasis multiplicity in TRAMP mice.

Identification of agents that are nontoxic but can delay onset and/or progression of prostate cancer, which is the second leading cause of cancer-related deaths among men in the United States, is highly desirable. We now show that p.o. gavage of garlic constituent diallyl trisulfide (DATS; 1 and 2 mg/day, thrice/week for 13 weeks beginning at age 8 weeks) significantly inhibits progression to poorly differentiated prostate carcinoma and pulmonary metastasis multiplicity in transgenic adenocarcinoma of mouse prostate (TRAMP) mice without any side effects. There was a trend of a decrease in average wet weights of the urogenital tract and prostate gland in 1 and 2 mg DATS-treated mice compared with controls ( approximately 25-46% decrease in DATS-treated mice compared with controls). The incidence and the area of the dorsolateral prostate occupied by the poorly differentiated carcinoma were significantly lower in both 1 and 2 mg DATS-treated mice compared with control mice. In addition, DATS administration resulted in a statistically significant decrease in pulmonary metastasis multiplicity compared with controls (P = 0.002). The dorsolateral prostate from DATS-treated TRAMP mice exhibited decreased cellular proliferation in association with induction of cyclinB1 and securin protein levels, and suppression of the expression of neuroendocrine marker synaptophysin. However, DATS administration did not have any appreciable effect on apoptosis induction, angiogenesis, or natural killer and dendritic cell function. In conclusion, the results of the present study show, for the first time, that DATS administration prevents progression to invasive carcinoma and lung metastasis in TRAMP mice.

Erythropoietin and normal brain development: receptor expression determines multi-tissue response.

Erythropoietin (EPO) is a hypoxia-inducible hormone required for erythroid differentiation. Expression of the EPO receptor is not restricted to hematopoietic cells and exhibits a multi-tissue distribution that includes neural cells, vascular endothelium and muscle progenitor cells. The ability for EPO to stimulate progenitor cell proliferation and prevent apoptosis is critical for maintenance of the erythroid lineage, but is also observed in neural and muscle progenitor cells. Mice lacking the EPO receptor die in utero due to severe anemia. However, even prior to lack of erythroid cell production in the embryo proper, these mice exhibit increased apoptosis in the brain as early as E10.5 and a reduction in the number of neural progenitor cells. Corresponding cultures of primary neural cells exhibit decreased neuron generation and increased sensitivity to reduced oxygen tension, and neurons do not survive after 24 h at low oxygen tension. In contrast, hypoxia induces EPO and EPO receptor in wild-type neuronal cells, and EPO enhances neuron survival at low oxygen tension. In vivo EPO is neuroprotective in adult animal models for brain ischemia. Induction of EPO and its receptor by hypoxia likely contributes to its neuroprotective activity and selective cell survival in the brain during hypoxic stress.