Circadian Responses to Chemo

The Scientist 21 April 2015 After exposure to curcumin, rat cancer

cell populations undergo a daily cycle of cell death. Like tissues throughout the body, tumors may also keep time, and a few studies have suggested that responses to cancer therapies may be stronger at particular times during the day. Related to this idea of “chronotherapy,” scientists presented unpublished data at the American Association for Cancer Research (AACR) meeting in Philadelphia showing that the effect of curcumin on rat glioblastoma cells cycles according to a circadian rhythm. Curcumin, a component of the spice turmeric, is known to have anti-cancer properties, and researchers are testing it out as a potential therapeutic agent. Given that curcumin can activate a gene important to regulating the circadian clock, Ashapurna Sarma, a graduate student in Michael Geusz’s lab at Bowling Green State University, along with colleagues at the University of Findlay’s College of Pharmacy in Ohio, wanted to see whether the anti-tumor effects of curcumin display any circadian rhythms. The team exposed rat glioblastoma cells to curcumin and filmed them for five days via time-lapse microscopy. The researchers then went back through the images and at five-minute intervals, manually counted the cancer cells. They found that “there’s a lot more apoptosis” in the treated cells compared to controls, Sarma told The Scientist. And for one particular concentration of curcumin, treated cells peaked in die-offs just about every 24 hours. “Curcumin is most effective at inducing apoptosis of glioma cells at a specific phase of the circadian cycle,” the authors wrote in their AACR poster. Sarma said the findings could have implications as researchers working to develop curcumin as a therapy for cancer patients; perhaps tweaking the timing of treatment could optimize its efficacy.

Metalloestrogens

The world abounds with substances that affect hormone balance. It is widely accepted that that there are many organic substances that affect hormone activity in the body. For example, soy has estrogen-like qualities and ginseng acts as an adrenal adaptogen. We can use these organic substances in ways that are beneficial to the body. On the other hand, organic substances like phthalates, PCBs and BPA mimic estrogens in a negative way. They create a burden of too much estrogen activity in the body. Researchers are now turning to inorganic sources of hormone disruption. In 2006, Darbre published a paper that identified many metals that have an effect on estrogen receptors. These effects include: altering gene expression, estrogenic activity, and displacing estrogens from estrogen receptors. Metals that have been identified to exert an influence on estrogen receptors include aluminum, antimony, arsenite, barium, cadmium, chromium, cobalt, copper, lead, mercury, nickel, selenite, tin, and vanadate. These metals are referred to as metalloestrogens. The paper also identified metalloestrogen activity at progesterone, testosterone and glucocorticoid (hydrocortisone-like) receptors. For example, cadmium has been shown to produce testosterone activity, displace testosterone from testosterone receptors and affect gene changes. Darbre concludes that all steroid hormone receptors may be affected. Some metalloestrogens are essential minerals. Cobalt, chromium, copper, and nickel are all needed in trace amounts for normal human body function. When the amounts of these minerals exceed the amount needed by the body, they begin to interfere with the hormone receptors. Other metalloestrogens, such as cadmium, aluminum, and lead, are not needed by the body in any amount. Cadmium is problematic because the human body doesn’t have an enzyme system to eliminate it once we are exposed. Making matters worse, the kidneys reabsorb cadmium rather than eliminate it. The amount of cadmium in our bodies continues to increase as we age. Common sources of cadmium, outside of industrial uses, include cigarette smoke and certain foods due to cadmium pollution in our environment. Cadmium may be a trigger for endometriosis. Several studies point to an association between the presence of cadmium in the body and endometrial tissue proliferation. One especially interesting study found melatonin was able to block the estrogenic effect of cadmium in endometrial tissue. Lead acts as a metalloestrogen by occupying hormone receptors. Unsurprisingly, lead contributes to a number of health conditions. The Environmental Protection Agency (EPA) recognizes lead can cause a number of problems for women. After menopause, increased levels of lead in the body increase one’s risk for hypertension, atherosclerosis, reduced kidney function, and decreased cognitive functioning with symptoms similar to dementia. Osteoporosis, another condition common in menopause, can cause lead to be released into the body as bone breaks down. Those of us who have been exposed to lead paints and leaded gasoline have higher levels of lead in our bones, causing higher levels of lead in our bodies as lead is released from our bones. To read more about lead, click this link: http://www.womensinternational.com/newsletter/article_GetTheLeadOut.html. As research expands and we dig deeper into hormone balance issues, more information emerges to help us solve the riddle of hormone imbalance. The effects of metalloestrogens are not always considered by busy practitioners. Furthermore, many are not trained to help reduce the body’s load of metalloestrogens. Two medical groups that focus on reducing metalloestrogens in the body are the International College of Integrated Medicine (www.icimed.com) and the American College for Advancement in Medicine (www.acam.org). References: Darbre, P.D. ‘Metalloestrogens: An Emerging Class of Inorganic Xenoestrogens With Potential to Add to the Oestrogenic Burden of the Human Breast.’ Journal of Applied Toxicology 2006; DOI:10.1002/jat.1135. http://epa.gov/aging/factsheets/weh-rd.html Silva N, Tennekoon K, Senanayake H, Samarakoon S. Metalloestrogen cadmium stimulates proliferation of stromal cells derived from the eutopic endometrium of women with endometriosis.Taiwan J Obstet Gynecol. 2013 Dec;52(4):540-5. L.W. Jackson, M.D. Zullo and J.M. Goldberg The association between heavy metals, endometriosis and uterine myomas among premenopausal women: National Health and Nutrition Examination Survey 1999 – 2002 Human Reproduction Vol 23 No. 3 pp 679-687, 2008 http://chemicaloftheday.squarespace.com/most-controversial/2014/9/4/metalloestrogens.html

Phellinus Linteus Extract Induces Autophagy and Synergizes With 5-Fluorouracil to Inhibit Breast Cancer Cell Growth

ABSTRACT
Phellinus linteus (PL) is a medicinal mushroom due to its several biological properties, including anticancer activity. However, the mechanisms of its anticancer effect remain to be elucidated. We evaluated the inhibitory effects of the ethanolic extract from the PL combined with 5-FU on MDA-MB-231 breast cancer cell line and to determine the mechanism of cell death. Individually, PL extract and 5-FU significantly inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner. PL extract (30 mg/mL) in combination with 5-FU (10 ?g/mL) synergistically inhibited MDA-MB-231 cells by 1.8-fold. PL did not induce apoptosis, as demonstrated by the DNA fragmentation assay, the sub-G1 population, and staining with annexin V-FITC and propidium iodide. The exposure of MDA-MB-231 cells to PL extracts resulted in several confirmed characteristics of autophagy, including the appearance of autophagic vacuoles revealed by monodansylcadaverine staining, the formation of acidic vesicular organelles, autophagosome membrane association of microtubule-associated protein light chain 3 (LC3) characterized by cleavage of LC3 and its punctuate redistribution, and ultrastructural observation of autophagic vacuoles by transmission electron microscopy. We concluded that PL extracts synergized with low doses of 5-FU to inhibit triple-negative breast cancer cell growth and demonstrated that PL extract can induce autophagy-related cell death.

INTRODUCTION
Metastasis is the main cause of therapeutic failure and death for breast cancer patients. To improve the prognosis of these patients, adjuvant chemotherapy is often used in a variety of clinical situations. However, the toxicity of these chemotherapeutic agents to normal tissues has been a major obstacle to successful cancer treatment. The antimetabolite 5-fluorouracil (5-FU) is a major chemotherapeutic agent for breast cancer treatment (1), and the mechanism of its cytotoxicity is the misincorporation of fluoronucleotides into DNA and RNA, thus inhibiting the normal function of these nucleic acids. Therefore, the development of less toxic and more effective anticancer drugs that can be used in combination with existing therapeutic agents may enhance treatment outcomes and reduce the toxicity of the existing drugs. Fungi have been intensively investigated as anticancer agents because they are able to modulate the immune responses against cancers with very low toxic potential (2) and thus represent a potentially important new source of anticancer agents.

Phellinus linteus (PL), a basidiomycete fungus, is commonly called Sangwhang in Taiwan and has gained significant recognition as a medicinal mushroom in traditional Oriental medicine (3). Studies have demonstrated that the extracts from the fruiting bodies or mycelium of PL not only stimulate immune function but also suppress tumor growth and metastasis in vitro (3–6). In vivo studies have also demonstrated that PL extracts can cause tumor regression (7). However, the mechanisms of its anticancer effect remain to be elucidated. Several studies have demonstrated that PL inhibits the metastasis of melanoma cells in mice through the regulation of urokinase-type plasminogen activator (8) and suppresses the growth of lung, prostate, and colon cancer cells by inducing cell cycle arrest and apoptosis (9–13). PL was also demonstrated to suppress the growth, angiogenesis, and invasion through the inhibition of AKT phosphorylation in breast cancer cells (14) and through the inhibition of Wnt/?-catenin signaling in colon cancer cells (15).

There are 3 reported cases of dramatically regressed cancers after treatment with PL, including 2 cases from Japan: 1 hormone-refractory prostate cancer with bone metastasis (16) and 1 hepatocellular carcinoma with multiple lung metastases (17); the third case occurred in Korea and was a hepatocellular carcinoma with skull metastasis (18). All of these cases suggested a linear relationship between the usage of PL and tumor regression. In addition, PL inhibited the growth of various prostate cancer cell lines without toxic effects on normal prostate epithelial cells (10) and reduced tumor growth and pulmonary metastasis without toxic effects in mice (6). Moreover, PL have been shown to synergize with doxorubicin in its noncytotoxic dose range to induce apoptosis in prostate and lung cancer cells (9,10), suggesting that PL can also function as an adjunct in cancer treatment to reduce the doses of conventional chemotherapeutic drugs and limit cytotoxicity.

Anticancer therapeutics activate several signal transduction pathways that regulate programmed cell death in cancer cells. Understanding the mechanisms of programmed cell death and designing specific therapeutic approaches to induce cell death in cancer cells are critical for cancer treatment (19). There are 2 morphologically distinct forms of programmed cell death: apoptosis and autophagic cell death. Traditional cancer therapies primarily aim to enhance apoptosis. However, cancer cells are often deficient in the induction of apoptosis, which results in resistance to most anticancer therapies (19,20). Thus, understanding the regulation and significance of the nonapoptotic form of programmed cell death in cancer therapy is critical to optimizing cancer therapy. Autophagic cell death is characterized by the massive degradation of essential organelles such as mitochondria. These intracellular contents are sequestered in a membrane-bound vesicle known as an autophagosome and then degraded following lysosomal fusion (21–23). Evidences indicate that autophagy plays a significant role in cancer initiation and progression. Nearly all therapeutic modalities currently used in cancer therapy, including cytotoxic chemotherapy, radiation, kinase inhibitors, and hormone therapy, can induce autophagy in cancer cells (21–23). Multiple studies have demonstrated that there is molecular cross-talk between autophagy and apoptosis (24), that the same stimulus can simultaneously induce both apoptosis and autophagy (25), and that 2 processes can be mutually exclusive, with each acting as a backup for the other (23).

PL has been shown to have anticancer effects in vitro and in vivo (3–7), but the underlying mechanism remained to be elucidated. PL was reported to induce apoptosis in various types of cancer, including colon, lung, prostate and melanoma cells (9–13,34). However, our study revealed that PL did not induce apoptosis detected by the DNA fragmentation assay, sub-G1 population, and annexin V-FITC/PI double staining in breast cancer cells.

Collins et al. showed that PL and the anti-cancer drug doxorubicin (Dox) did not induce apoptosis in prostate cancer cells at relatively low doses; however, the combination treatment with low doses of PL and Dox resulted in a synergistic effect on the induction of apoptosis (10). Guo et al. demonstrated that PL modulated cell cycle arrest at a low dose and induced apoptosis at a high dose in lung cancer cells (9). Taken together, the dose of PL used in this study may be the major reason for the differences from the previous studies. In addition, the mycelial species, extraction method (hot water vs. ethanolic), culture conditions and cell lines may affect the results.

Source
Lee W-y, Hsu K-F, Chiang T-A. Nutrition and Cancer. Volume 67, Issue 2, 2015. DOI: 10.1080/01635581.2015.989374
References
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2. Sullivan R, Smith JE, and Rowan NJ: Medicinal mushrooms and cancer therapy: translating a traditional practice into Western medicine. Perspect Biol Med 49, 159–170, 2006.
3. Zhu T, Kim SH, and Chen CY: A medicinal mushroom: Phellinus linteus. Curr Med Chem 15, 1330–1335, 2008.
4. Kim GY, Lee JY, Lee JO, Ryu CH, Choi BT, : Partial characterization and immunostimulatory effect of a novel polysaccharide-protein complex extracted from Phellinus linteus. Biosci Biotechnol Biochem 70, 1218–1226, 2006.
5. Han SB, Lee CW, Kang JS, Yoon YD, Lee KH, : Acidic polysaccharide from Phellinus linteus inhibits melanoma cell metastasis by blocking cell adhesion and invasion. Int Immunopharmacol 6, 697–702, 2006.
6. Han SB, Lee CW, Jeon YJ, Hong ND, Yoo ID, : The inhibitory effect of polysaccharides isolated from Phellinus linteus on tumor growth and metastasis. Immunopharmacology 41, 157–164, 1999.
7. Tsuji T, Du W, Nishioka T, Chen L, Yamamoto D, : Phellinus linteus extract sensitizes advanced prostate cancer cells to apoptosis in athymic nude mice. PLoS One 5, e9885, 2010.
8. Lee HJ, Lee HJ, Lim ES, Ahn KS, Shim BS, : Cambodian Phellinus linteus inhibits experimental metastasis of melanoma cells in mice via regulation of urokinase type plasminogen activator. Biol Pharm Bull 28, 27–31, 2005.
9. Guo J, Zhu T, Collins L, Xiao ZX, Kim SH, : Modulation of lung cancer growth arrest and apoptosis by Phellinus Linteus. Mol Carcinog 46, 144–154, 2007.
10. Collins L, Zhu T, Guo J, Xiao ZJ, and Chen CY: Phellinus linteus sensitises apoptosis induced by doxorubicin in prostate cancer. Br J Cancer 95, 282–288, 2006.
11. Zhu T, Guo J, Collins L, Kelly J, Xiao ZJ, : Phellinus linteus activates different pathways to induce apoptosis in prostate cancer cells. Br J Cancer 96, 583–590, 2007.
12. Li G, Kim DH, Kim TD, Park BJ, Park HD, : Protein-bound polysaccharide from Phellinus linteus induces G2/M phase arrest and apoptosis in SW480 human colon cancer cells. Cancer Lett 216, 175–181, 2004.
13. Park HJ, Choi SY, Hong SM, Hwang SG, and Park DK: The ethyl acetate extract of Phellinus linteus grown on germinated brown rice induces G0/G1 cell cycle arrest and apoptosis in human colon carcinoma HT29 cells. Phytother Res 24, 1019–1026, 2010.
14. Sliva D, Jedinak A, Kawasaki J, Harvey K, and Slivova V: Phellinus linteus suppresses growth, angiogenesis and invasive behaviour of breast cancer cells through the inhibition of AKT signalling. Br J Cancer 98, 1348–1356, 2008.
15. Song KS, Li G, Kim JS, Jing K, Kim TD, : Protein-bound polysaccharide from Phellinus linteus inhibits tumor growth, invasion, and angiogenesis and alters Wnt/beta-catenin in SW480 human colon cancer cells. BMC Cancer 11, 307, 2011.
16. Shibata Y, Kurita S, Okugi H, and Yamanaka H: Dramatic remission of hormone refractory prostate cancer achieved with extract of the mushroom, Phellinus linteus. Urol Int 73, 188–190, 2004.
17. Nam SW, Han JY, Kim JI, Park SH, Cho SH, : Spontaneous regression of a large hepatocellular carcinoma with skull metastasis. J Gastroenterol Hepatol 20, 488–492, 2005. [CrossRef], [PubMed], [Web of Science ®]
18. Kojima H, Tanigawa N, Kariya S, Komemushi A, Shomura Y, : A case of spontaneous regression of hepatocellular carcinoma with multiple lung metastases. Radiat Med 24, 139–142, 2006.
19. Melet A, Song K, Bucur O, Jagani Z, Grassian AR, : Apoptotic pathways in tumor progression and therapy. Adv Exp Med Biol 615, 47–79, 2008.
20. Johnstone RW, Ruefli AA, and Lowe SW: Apoptosis: a link between cancer genetics and chemotherapy. Cell 108, 153–164, 2002.
21. Kondo Y and Kondo S: Autophagy and cancer therapy. Autophagy 2, 85–90, 2006.
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34. Park HJ, Han ES, and Park DK: The ethyl acetate extract of PGP (Phellinus linteus grown on Panax ginseng) suppresses B16F10 melanoma cell proliferation through inducing cellular differentiation and apoptosis. J Ethnopharmacol 132, 115–121, 2010.

Correlation between pretherapeutic d-dimer levels and response to neoadjuvant chemotherapy in patients with advanced esophageal cancer.

Source Tomimaru Y, Yano M, Takachi K, et al. Dis Esophagus. 2008;21(4):281-7. doi: 10.1111/j.1442-2050.2007.00758.x. Neoadjuvant chemotherapy may improve survival of responders in esophageal cancer patients but is useless and harmful in non-responders. Thus, it is important to predict the effect of the chemotherapy, and that any predictor must be applicable clinically. The aim of this study is to examine the correlation between pretherapeutic hypercoagulopathy as determined by plasma d-dimer levels and response to chemotherapy. In 71 patients with esophageal cancer who underwent neoadjuvant chemotherapy (cisplatin, adriamycin and 5-fluorouracil) followed by surgery, plasma d-dimer levels were measured before chemotherapy and the clinical and pathological responses to chemotherapy were assessed at 4 weeks after therapy (after surgery). Pretherapeutic plasma d-dimer level was significantly lower in clinical responders (complete response/partial response [CR/PR]; 0.62 +/- 1.10 microg/mL, mean +/- SD) than in non-responders (no change/progressive disease [NC/PD]; 1.15 +/- 1.08 microg/mL, P = 0.0491), and in pathological responders (Grade 1b-3; 0.62 +/- 1.11 microg/mL) and non-responders (Grade 0-1a; 1.15 +/- 1.05 microg/mL, P = 0.0107). The optimal cut-off level of the plasma d-dimer levels for predicting clinical and pathological responses was 0.6 microg/mL. Then, sensitivity and specificity for the prediction of CR/PR were 68% and 73%, and those for Grade 1b-3 were 91% and 69%, respectively. Our results suggested that pretherapeutic plasma d-dimer level correlated significantly with clinical and pathological responses to chemotherapy. Pretherapeutic plasma d-dimer level can be used as a predictor for chemosensitivity.

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC)

Guibitang, a traditional herbal medicine, induces apoptotic death in A431 cells by regulating the activities of mitogen-activated protein kinases

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are commonly referred to as non-melanoma skin cancers [1,2]. BCC is a slow-growing cancer that does not usually metastasize. Similarly, SCC is frequently localized without evidence of blood-born metastasis, making direct treatment of the tumor straightforward. However, SCC is the sixth most common cancer worldwide, and its incidence has increased dramatically at multiple sites in the body, including the head and neck, cervix, and lung [3,4]. Accordingly, it is necessary to develop novel effective chemopreventive agents to inhibit the development of SCC.

Guibitang (GBT), known as ‘Kihi-to’ in Japan and ‘Gui-Pi-Tang’ in China, is a traditional medicine and herbal formula that has been used for several hundred years, predominantly to treat insomnia, amnesia, palpitations, anxiety, fatigue, poor appetite, and depression [5]. Recent studies have reported the specific bioactivities of GBT, which include immune regulation [6], antioxidant effects [7], and protective effect of the gastric mucosa [8].

GBT is an aqueous polyherbal formulation that contains 12 herbs: Angelica gigas Nakai, Dimocarpus longan, Zizyphus jujuba Miller (seed), Polygala tenuifolia, Panax ginseng, Astragalus membranaceus, Atractylodes ovate, Poria cocos, Inula helenium, Glycyrrhiza glabra, Zingiber officinale, and Zizyphus jujuba Miller (Fructus). GBT also regulates chronic fatigue syndrome-associated cytokine production, whereas the addition of Gardenia jasminoides, Paeonia suffruticosa, and Bupleurum falcatum to GBT improves palliative care in patients undergoing chemotherapy for ovarian cancer [9]. Although it has been shown that adding several herbs to GBT results in anti-cancer effects against gynecological or lung cancer, the molecular mechanisms behind these effect of GBT remain unclear.

Tumorigenesis is caused by unregulated growth of cells resulting from DNA damage, mutations of functional genes, dysregulation of the cell cycle, and loss of apoptotic function [10]. Therefore, regulating the induction of apoptosis by modulating cell growth and survival-related signaling pathways is a common and major target for cancer therapies [11]. Among several signaling pathways in cancer cells, mitogen-activated protein kinase (MAPK) signals including extracellular signal-regulated kinases (ERK), p38 kinases, and c-Jun N-terminal kinases (JNK), take an important role in cell death and survival [12]. The regulation of ERK activation is induced by conditions of stress such as some agents and oxidant injury, which plays a major role in regulating cell growth and differentiation [13]. JNK and p38 are activated in response to several stress signals including tumor necrosis factor and hyperosmotic condition, which is associated with induction of apoptosis [14].

GBT showed cytotoxic activity against three different squamous cell carcinoma, especially on A431 cells. GBT induced the apoptosis through activating the caspase-8 in A431 cells. Inhibition of A431 cell growth by GBT was caused by G1-phase arrest through regulating proteins associated with cell cycle progression, such as cyclin D1, p21, and p27. Furthermore, GBT regulated the activation of mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK), and activated p53, a tumor suppressor protein. In MAPKs inhibitor study, inhibitors respectively blocked GBT-induced cell viability, indicating that MAPKs signals play critical role in cell death caused by GBT. In vivo xenografts, daily oral administration of 600 mg/kg GBT efficiently suppressed the tumorigenic growth of A431 cells without side effects such as loss of body weight and change of toxicological parameters compared to vehicle.

In the present study, Yim et al., (2014) evaluated whether GBT shows the anti-cancer effect in A431 human squamous carcinoma cells, which demonstrated that GBT induces apoptosis of cancer cells specifically, as an inhibition of the cell growth via regulating MAPK signaling pathway in A431 cells.

GBT decreases cell viability in A431 human squamous carcinoma cells
Six different human cancer cell lines (A431 [squamous], AGS [stomach], HeLa [cervical], Caki-1 [kidney], SK-Hep-1 [liver], and HCT116 [colon]) were treated with 500 ?g/mL GBT for 48 h, and cell viability was assessed by an MTT assay. Although most cell lines were unaffected, the viability of A431 cells was inhibited >35% by treatment with GBT (Figure 2A). Therefore, subsequent tests focused on A431 cells.

To further define the inhibitory action of GBT on SCCs, the suppression of cell growth by GBT on three different SCC lines (SCC12, SCC13, and A431) was evaluated. As shown in Figure 2B, treatment with 500 and 1000 ?g/mL GBT for 48 h reduced the viability of A431 cells by 35% and 52%, respectively. Treatment of SCC13 cells with 1000 ?g/mL GBT also inhibited the cell growth by ~30% although these effects were not as potent as those observed in A431 cells.

In contrast, the viability of SCC12 cells was not affected significantly by GBT. The potential cytotoxic effect of GBT on normal cells was assessed using normal human HaCaT keratinocytes and mouse primary liver cells. HaCaT cells were unaffected by GBT under the same conditions that were cytotoxic to A431 cells (Figure 2C). In addition, no cytotoxic effects on primary liver cells were observed by treatment with 500 ?g/mL or 1000 ?g/mL GBT. Instead, GBT weakly increased the viability of liver primary cells in a dose- and time-dependent manner. These results suggest that GBT has cancer-specific cytotoxic effect on A431 cells, without affecting normal cells.

Source
Yim N-H, Kim A, Liang C et al. BMC Complementary and Alternative Medicine 2014, 14:344 doi:10.1186/1472-6882-14-344

References
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