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How Much Emodin Does It Take To Start Decreasing Tumor Size?

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Emodin and rhein decrease levels of hypoxia-inducible factor-1α in human pancreatic cancer cells and attenuate cancer cachexia in athymic mice carrying these cells.
Emodin and rhein decrease levels of hypoxia-inducible factor-1α in human pancreatic cancer cells and attenuate cancer cachexia in athymic mice carrying these cells


Abstract

The transcription factor hypoxia-inducible factor-1 (HIF-1) consists of oxygen-sensitive HIF-1α and constitutive HIF-1β. HIF-1α is undetectable in normal cells, but cancer cells frequently express HIF-1α to support their growth, angiogenesis, and high glycolysis (also known as the Warburg effect). The Warburg effect in cancer cells increases energy expenditure and thus participates in cancer-induced metabolic disorder, cancer cachexia. In the present study, we investigated whether two components of Rheum palmatum, emodin and rhein, inhibited HIF-1α expression in human pancreatic cancer cells and whether the inhibiting effect, if any, attenuated cancer cachexia. Using Western blotting, we demonstrated that emodin and rhein decreased HIF-1α expression in MiaPaCa2 and four other human pancreatic cancer cell lines. We also examined HIF-1α expression when MiaPaCa2 cells were exposed to PX-478, noscapine, and phenethyl isothiocyanate, as these compounds were known to inhibit HIF-1α expression in different cancer cells. PX-478 and noscapine inhibited HIF-1α expression to a less extent than emodin and rhein, and phenethyl isothiocyanate did not inhibit HIF-1α expression in tested concentrations. We obtained evidence that emodin and rhein decreased HIF-1α by decreasing its biosynthesis but not gene transcription or protein stability. When MiaPaCa2 cells were implanted in athymic mice, emodin and rhein inhibited cancer-cell growth and HIF-1α expression. In these athymic mice, emodin and rhein also attenuated two pathological constituents of cancer cachexia, namely high hepatic gluconeogenesis and skeletal-muscle proteolysis. In conclusion, emodin and rhein decrease pancreatic cancer cell's growth and HIF-1α expression and attenuate cancer cachexia in the athymic mice carrying the cancer cells.

Normal mammalian cells produce energy primarily by oxidative phosphorylation, whereas cancer cells do it mainly by glycolysis even though glycolysis is of low efficiency in energy production. To produce enough energy, cancer cells over-express glucose transporters and glycolytic enzymes to raise glycolysis to high levels. In 1920s, Otto Warburg first described the high glycolysis in cancer cells, so the phenomenon is also called the Warburg effect [1]. How cancer cells manage to over-express glucose transporters and glycolytic enzymes had been unknown, until a transcription factor namely hypoxia-inducible factor-1 (HIF-1) was discovered in 1992 [2].

HIF-1 consists of oxygen-sensitive HIF-1α and constitutive HIF-1β. After HIF-1α is synthesized, its P402and P564 residues are hydroxylated by oxygen and prolyl hydroxylase domain proteins-1, -2, and -3 (PHDs-1, -2, and -3). Subsequently, hydroxylated HIF-1α is associated with the Von Hippel-Lindau protein, tagged with ubiquitin, and degraded in proteasomes [3, 4]. Thus, normal mammalian cells have HIF-1β but not HIF-1α. When cells are stressed by hypoxia, HIF-1α is saved from degradation and associated with HIF-1β. The resulting HIF-1 transcriptionally up-regulates its target genes the proteins encoded by which include glucose transporters, glycolytic enzymes, and growth- and angiogenic factors. Thus, when hypoxic cells express HIF-1α, their glycolysis, viability, and angiogenesis increase, so they are more resistant to hypoxic stresses [5].

Cancer cells frequently express HIF-1α and thereby acquire increased glycolysis, viability, and angiogenesis [5, 6]. The mechanisms of cancer-induced HIF-1α are multifactorial, including a decrease in HIF-1α degradation that is associated with intra-tumor hypoxia and an increase in HIF-1α production that is associated with oncogene expression [7, 8]. If HIF-1α expression is inhibited in cancer cells, aggressive behaviors decrease in these cells.

Cancer cachexia is a cancer-induced metabolic syndrome whose pathological constituents include increased energy expenditure, augmented hepatic gluconeogenesis, uncontrolled skeletal-muscle wasting (proteolysis), and unrestrained fat lipolysis [9, 10]. When these pathologies persist, body weight decreases. It remains unclear how these pathologies are initiated, so cancer cachexia is still treated with palliative measures.

Due to the Warburg effect, cancer cells consume a good deal of blood glucose and release lactate as the waste. As a result, hepatic gluconeogenesis increases to recycle tumor-produced lactate to glucose. When the glucose is released to the blood, cancer cells may take it up for glycolysis again. In cancer patients, the glucose-lactate shuttle cannot meet the demand for glucose. Thus, skeletal muscle and adipose tissues undergo catabolic metabolisms to release more glucose precursors for the liver. Consequently, body weight decreases. In this light, the Warburg effect in cancer cells is a triggering event in the pathogenesis of cancer cachexia. Thus, if the Warburg effect decreases following an inhibition of cancer-induced HIF-1α, cancer cachexia may be reversed at least to some extent.

Numerous compounds are reported to inhibit HIF-1α expression, but no HIF-1α inhibitors are clinically available as anti-cancer drugs [1116]. We undertook the present study to investigate whether emodin and rhein from Rheum palmatum inhibited HIF-1α expression in human pancreatic cancer cells. We also investigated whether the HIF-1α-inhibiting effect of emodin and rhein, if any, attenuated cancer cachexia in the athymic mice that carried the cancer cells.

RESULTS

The effects of emodin and rhein on HIF-1α in pancreatic cancer cells

When five human pancreatic cancer cell lines were exposed to emodin or rhein, HIF-1α was decreased dose-dependently by either reagent (Figure1A--1E). In these cell lines, the lowest effective doses of emodin were varied from 50 μM to 100 μM and those of rhein from 20 μM to 50 μM. When five cell lines were exposed to 200μM emodin, their HIF-1α expression levels equaled to 24%-55% of the control value (Figure1A--1E). When five cell lines were exposed to 200μM rhein, their HIF-1α expression levels equaled to 28%-50% of the control value (Figure1A1A--1E). These results demonstrate that both emodin and rhein inhibited HIF-1α expression in pancreatic cancer cells.

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The effects of test reagents on HIF-1α expression

Test reagents were diluted in culture media to give the final concentrations of 10, 20, 50, 100, and 200 μM. These concentrations are denoted by 1, 2, 3, 4 and 5, respectively, in this figure. Pancreatic cancer cells were incubated in hypoxia for 6h, using the reagent-containing media. Control cells (ctr.) were incubated without tested reagents. HIF-1α was determined by Western blot, using β-actin for loading control. The upper blots in each panel are representative results for 5-7 experiments, and the lower histograms summarize HIF-1α-actin ratios in all the experiments. * P<0.05, ** P<0.01, and *** P<0.001, compared to control values. (A-E). The effects of emodin and rhein on HIF-1α expression in AsPC-1 (A), BxPC-3 (B), HPAF-2 (C), MiaPaCa2 (D), and Panc-1 (E) pancreatic cancer cells. (F-H). The effects of PX-478 (F), noscapine (G), and phenethyl isothiocyanate (H) on HIF-1α expression in MiaPaCa2 cells.


Two signaling pathways, which include Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) respectively, induce HIF-1α expression by increasing HIF-1α biosynthesis [8, 11, 15, 1720]. The proximal ends of these pathways start from growth-factor receptors on cellular surface. After these receptors are activated, the signal is passed forward when kinases in the signaling pathways are phosphorylated one after another [8]. Emodin and rhein decreased phosphorylated Akt and ERK/1/2 (p-Akt and p-ERK1/2) without changing total Akt and ERK1/2 (Figure (Figure2B).2B). This result suggests that emodin and rhein decrease levels of HIF-1α by down-regulating the intracellular signaling pathways that lead to the expression of HIF-1α.

When mammalian cells are in normoxia, HIF-1α is hydroxylated by PHDs and O2 before it is degraded in proteasomes [3, 4]. If proteasome activity is inhibited by MG-132, hydroxylated HIF-1α is stable even in normoxia [21]. In the present study, emodin and rhein decreased hydroxylated HIF-1α in normoxic MiaPaCa2 cells in the presence of MG-132 (Figure3A). Thus, emodin and rhein decreased HIF-1α biosynthesis in both hypoxia and normoxia. HIF-1α mRNA increased when MiaPaCa2 cells were exposed to emodin and rhein ( (Figure3B). It may reflect a feedback when the protein of HIF-1α was decreased by emodin and rhein.

Next, we investigated whether emodin and rhein decreased HIF-1α stability. When cells are in hypoxia, their HIF-1α degradation may not be inhibited totally. If HIF-1α production is inhibited in hypoxic cells, their HIF-1α contents will decrease overtime as in normoxic cells [21]. To study HIF-1α degradation in hypoxic conditions, we incubated MiaPaCa2 cells in hypoxia for 6h without or with emodin (200 μM) or rhein (200 μM). Then, CHX (cycloheximide) was added to culture media (100 μg/ml) to inhibit HIF-1α production, and the cells were further incubated in hypoxia for 10, 30, and 60 minutes. By examining HIF-1α contents at these time points, we appreciated the way HIF-1α was degraded in the absence or presence of emodin and rhein. After CHX was added, the contents of HIF-1α were decreased gradually in control cells (Figure (Figure3C).3C). As expected, HIF-1α expression was decreased by emodin or rhein before the start (Min-0) of the degrading period (Figure3C). After CHX was added, however, the way HIF-1α was degraded in the emodin- or rhein-treated cells was similar to that seen in the control cells (Figure3C and 3D). The data suggest that the inhibition of HIF-1α expression induced by emodin and rhein was not a result of increased HIF-1α degradation. In separate experiments, we determined PHD-2 protein and mRNA in the presence and absence of emodin and rhein. Emodin decreased PHD-2 expression at both protein and mRNA levels, and rhein decreased PHD-2 protein (Figure3E and3F). These data may reflect a feedback regulation in PHD-2 expression when HIF-1α, as a substrate of PHD-2, was decreased by emodin and rhein.

The effects of emodin and rhein on tumor-carrier's energy homeostasis

Hepatic gluconeogenesis starts when pyruvate carboxylase (PCB) carboxylates pyruvate and ends when glucose-6-phosphatase (G-6-Pase) hydrolyzes glucose-6-phosphate to give glucose. In the present study, hepatic G-6-Pase increased when tumor-carrying mice were only treated with vehicle (Figure (Figure6A).6A). It indicates that hepatic gluconeogenesis increased when mice carried MiaPaCa2 cells. When tumor carriers were treated with emodin or rhein, hepatic G-6-Pase expression was normal. The data suggest that cancer-induced hepatic gluconeogenesis was attenuated when HIF-1α expression in cancer cells was decreased by emodin and rhein. Hepatic PCB expression tended to increase in tumor carriers, but the change was not significant statistically (Figure (Figure6A).6A). All tumor-carrying groups showed significant decrease in hepatic glycogen, compared to the control value, and no significant difference was found between any tumor-carrying groups (Figure (Figure6B).6B). Thus, cancer-induced decrease in hepatic glycogen cannot be relieved by emodin and rhein.

Emodin and rhein decreased tumor volume and weight, which suggests that both drugs inhibited cancer-cell growth. When tumor section was analyzed, emodin and rhein decreased both total section area and the section area that was occupied by healthy tissue. Neither emodin nor rhein changed the section area that was occupied by dead cells. Thus, emodin and rhein may inhibit tumor growth by decreasing cell proliferation rather than promoting cell death.

In conclusion, emodin and rhein inhibited HIF-1α expression in human pancreatic cancer cells both in vitro and in vivo. The induced inhibition of HIF-1α expression was associated not only with a decrease in cancer cell's growth but also with an improvement in cancer-induced hepatic gluconeogenesis and skeletal-muscle wasting. Emodin and rhein may be used as anticancer drugs that both sabotage malignant tumor cells and improve energy homeostasis in the tumor carrier.

Implantation of MiaPaC2 cells in athymic mice (in vivo)

Male athymic Balb/c mice were bought from Hua-Fu-Kang Bioscience (Beijing, China). When arriving at our hospital, they were 4-5 weeks old and weighed 17-20 g. We followed the guide for the care and use of laboratory animals, 8th edition (NIH, 2011). After acclimation, mice were designated to three groups (10 mice per group), and 3 × 106 MiaPaCa2 cells were injected subcutaneously in each mouse. Afterwards, all mice were kept for 8 weeks. In the last 4 weeks, emodin and rhein were dissolved in PBS (phosphate buffered solution) and administered by gavage (50 mg/kg) [Liquid compounds may be administered directly into the stomach of mice and rats via a technique called oral gavage. In this procedure a stainless steel bulb tipped gavage needle or a flexible cannula or tube is attached to a syringe and used to deliver the compound into the stomach.] in two groups, respectively. Vehicle was administered by gavage in the last group. All gavage treatments were given once a day and five days a week. A group of intact athymic mice (n = 5) were also included. When mice were sacrificed, they were anesthetized using 5% chloral hydrate and exsanguinated in the orbital sinus. Blood was collected and centrifuged (1,500 g, 10 min) to obtain plasma. Subcutaneous tumor and inguinal fat pads were removed surgically, and so was the skeletal muscle in hind legs. The abdominal cavity was opened. The liver and epididymal fat pads were removed.

*** Human Equivalent Dose= Mouse Dose/12.3 = 50/12.3 = 4.065mg/kg
*** So for a 70kg human, that's ~284mg/Day
 

aguilaroja

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Jul 24, 2013
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Emodin and rhein decrease levels of hypoxia-inducible factor-1α in human pancreatic cancer cells and attenuate cancer cachexia in athymic mice carrying these cells
Due to the Warburg effect, cancer cells consume a good deal of blood glucose and release lactate as the waste. As a result, hepatic gluconeogenesis increases to recycle tumor-produced lactate to glucose. When the glucose is released to the blood, cancer cells may take it up for glycolysis again. In cancer patients, the glucose-lactate shuttle cannot meet the demand for glucose. Thus, skeletal muscle and adipose tissues undergo catabolic metabolisms to release more glucose precursors for the liver. Consequently, body weight decreases. In this light, the Warburg effect in cancer cells is a triggering event in the pathogenesis of cancer cachexia. Thus, if the Warburg effect decreases following an inhibition of cancer-induced HIF-1α, cancer cachexia may be reversed at least to some extent.

...Emodin and rhein decreased tumor volume and weight, which suggests that both drugs inhibited cancer-cell growth. When tumor section was analyzed, emodin and rhein decreased both total section area and the section area that was occupied by healthy tissue. Neither emodin nor rhein changed the section area that was occupied by dead cells. Thus, emodin and rhein may inhibit tumor growth by decreasing cell proliferation rather than promoting cell death.

In conclusion, emodin and rhein inhibited HIF-1α expression in human pancreatic cancer cells both in vitro and in vivo.

Thank you. There are many important recent findings about emodin.
I am guessing in this study, the five times per week dosing choice was based on lab worker schedule, not theory. An effective tumor-reducing substance should work on the weekend, too.

Going by figures 4E and 4F, tumor weight and volume shrunk roughly 40%. Human pancreatic cancer has poor outcome probability generally. If this were a pharmaceutical instead of an herbal derivative, the same results would be shouted in business PR.
 

Light

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Going by figures 4E and 4F, tumor weight and volume shrunk roughly 40%. Human pancreatic cancer has poor outcome probability generally. If this were a pharmaceutical instead of an herbal derivative, the same results would be shouted in business PR.
Thanks for adding that, the images didn't appear right in my post for some reason.
 

joaquin

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