Heme oxygenase (HO)-1

As positive controls, PGJ2 and Hemin significantly elevated HMOX activity and expression after 4 and 12 hours, respectively. Indeed, after 48 hours, it was discovered that 30 μM heme significantly and dose-dependently affected the proliferation of all the cell lines examined. Hemin therapy resulted in 62±5%, 51±3%, and 38±8%, respectively, reductions in the proliferation of PA-TU-8902, BxPC-3, and MiaPaCa-2 cancer cells, with p<0.0001 for all comparisons. Moreover, Hemin saw an increased antiproliferative impact of statins, which was recorded as a reduction in cell division following a 48-hour co-treatment period [1].

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Methods: In vitro effects of various statins and Hemin, a heme oxygenase inducer, on cell proliferation were evaluated in PA-TU-8902, MiaPaCa-2 and BxPC-3 human pancreatic cancer cell lines. The effect of statins on heme oxygenase activity was assessed and heme oxygenase-silenced cells were used for pancreatic cancer cell proliferation studies. Cell death rate and reactive oxygen species production were measured in PA-TU-8902 cells, followed by evaluation of the effect of cerivastatin on GFP-K-Ras trafficking and expression of markers of invasiveness, osteopontin (SPP1) and SOX2.
Results: While simvastatin and cerivastatin displayed major anti-proliferative properties in all cell lines tested, pravastatin did not affect the cell growth at all. Strong anti-proliferative effect was observed also for hemin. Co-treatment of cerivastatin and Hemin increased anti-proliferative potential of these agents, via increased production of reactive oxygen species and cell death compared to individual treatment. Heme oxygenase silencing did not prevent pancreatic cancer cells from the tumor-suppressive effect of cerivastatin or hemin. Cerivastatin, but not pravastatin, protected Ras protein from trafficking to the cell membrane and significantly reduced expressions of SPP1 (p < 0.05) and SOX2 (p < 0.01).
Conclusions: Anti-proliferative effects of statins and Hemin on human pancreatic cancer cell lines do not seem to be related to the heme oxygenase pathway. While hemin triggers reactive oxygen species-induced cell death, cerivastatin targets Ras protein trafficking and affects markers of invasiveness [1].

The renal cortex's HO-1 levels started to progressively rise following an intraperitoneal infusion of heme (100 μmol/kg). 24 hours following Hemin pretreatment, HO-1 levels rose. Renal tubules are where HO-1 is primarily expressed. Following Hemin pretreatment, there is no change in the kidneys' HO-2 levels [2].

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Heme oxygenase (HO)-1 confers transient resistance against oxidative damage, including renal ischemia-reperfusion injury (IRI). We investigated the potential protective effect of HO-1 induction in a mouse model of renal IRI induced by bilateral clamping of the kidney arteries. The mice were randomly assigned to five groups to receive an intraperitoneal injection of PBS, Hemin (an HO-1 inducer, 100μmol/kg), Hemin+ZnPP (an HO-1 inhibitor, 5mg/kg), Hemin+PD98059 (a MEK-ERK inhibitor, 10mg/kg) or a sham operation. All of the groups except for the sham-operated group underwent 25min of ischemia and 24 to 72h of reperfusion. Renal function and tubular damage were assessed in the mice that received hemin or the vehicle treatment prior to IRI. The renal injury score and HO-1 protein levels were evaluated via H&E and immunohistochemistry staining. Hemin-preconditioned mice exhibited preserved renal cell function (BUN: 40±2mg/dl, creatinine: 0.53±0.06mg/dl), and the tubular injury score at 72h (1.65±0.12) indicated that tubular damage was prevented. Induction of HO-1 induced the phosphorylation of extracellular signal-regulated kinases (ERK) 1/2. However, these effects were abolished with ZnPP treatment. Kidney function (BUN: 176±49mg/dl, creatinine: 1.54±0.39mg/dl) increased, and the tubular injury score (3.73±0.09) indicated that tubular damage also increased with ZnPP treatment. HO-1-induced tubular epithelial proliferation was attenuated by PD98059. Our findings suggest that HO-1 preconditioning promotes ERK1/2 phosphorylation and enhances tubular recovery, which subsequently prevents further renal injury[2].

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Hemin preconditioning induces HO-1 expression in the kidney cortex [2]
Following the i.p. administration of Hemin (100 μmol/kg), the HO-1 level in the renal cortex began to increase gradually. The HO-1 level reached its peak 24 h after Hemin preconditioning. HO-1 was expressed mainly in the renal tubules (Fig. 1a, b). The HO-2 level in the kidney did not change following hemin preconditioning.

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Hemin preconditioning enhances early kidney function recovery following kidney IRI [2]
Eight hours after Hemin preconditioning, the mice were subjected to bilateral renal artery clamping for 25 min. Both the vehicle and Hemin-preconditioned groups exhibited tubular dilatation, necrosis, brush border loss, and sloughing of non-necrotic tubular cells in the tubular lumen 24 h after IRI. The tubular injury score of the hemin-preconditioned group was slightly lower than the score of the vehicle group 24 h after IRI. However, the vehicle and hemin-preconditioned groups showed the same level of injury to kidney function 24 h after IRI (Fig. 2). Two-way ANOVA was conducted to examine the effects of preconditioning and time and to investigate the interactions between BUN, creatinine, and the tubular injury score (for preconditioning and condition effects). We detected significant effects when assessing the time effect (0, 24, and 72 h), the preconditioning effect (sham, vehicle, and hemin groups), and the interactions between time and preconditioning for all of the tests.

HMOX activity measurement [1]
Cells in plates were treated with statins and Hemin. After 12 h, cells were washed twice with ice-cold phosphate buffer and finally collected into freshly added phosphate buffer and centrifuged. The pellet was resuspended in 150 μl of 0.1 M potassium phosphate buffer (pH = 7.4) and sonicated with an ultrasonic cell disruptor. The protein concentration was assessed using the DC™ Protein Assay according to the manufacturer's instruction. A total of 0.15 mg of protein was incubated for 15 min at 37 °C in CO-free septum-sealed vials containing 20 μl of 4.5 mM NADPH as previously described. The amount of CO generated by HMOX activity was quantified by gas chromatography with a reduction gas analyzer and calculated as pmol CO/h/mg protein. Five μM PGJ2 was used as a positive control of heme regulation. Results were expressed as % of control.

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Ras protein translocation assay [1]
PA-TU-8902 cells were seeded in dishes with glass bottom 6 h before transfection by pEGFP-KrasWT (GFP – green fluorescent protein, WT-wild type) plasmid prepared as described previously. Transfection was carried out using FuGene HD according to the manufacturer’s instructions. Cerivastatin (12 μM), pravastatin (12 μM) and Hemin (30 μM) were added 12 h post transfection and the cells incubated with the agents for 24 h. Intracellular localization of the GFP-K-Ras protein was visualized by confocal microscopy, using a spinning disk confocal microscope equipped with solid state laser (488 nm for continual excitation). Emission was collected through a single-band filter. The images were obtained and analyzed with the iQ2 software.

Cell proliferation assay [1]
For the cell proliferation assay, cells were seeded into 96 well (5–12.5 x 104 cells per ml according to the cell line) and kept at 37 °C and 5 % CO2. After 24 h, cells were treated with statins or/and Hemin, followed by the MTT test as a general cell proliferation assay. As we experienced difficulties with Hemin-treated samples using MTT test due to interfering effects of hemin, we further used the more sensitive CellTiter-Glo Luminescent Cell Viability Assay. Both tests were used according to the manufacturer's instructions. Results were expressed as % of controls.

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Western blot analyses [1]
For protein expression analyses, cells were transfected with esiRNA universal control or esiRNA HMOX1/2 as mentioned previously. After 24 h, cells were treated with 30 μm Hemin for 20 h. Hemin treatment was used to upregulate HMOX1 protein expression to cumulate detectable levels of HMOX1 protein. Thirty μg of total protein were separated on 12 % polyacrylamide gel and then transferred to nitrocellulose membrane. After blocking in Tween-PBS with 5 % milk for at least 1 h, membranes were incubated with HMOX1 antibody (1:1000), or β-actin (1:1000) overnight at 4 °C. After washing, membranes were incubated with anti-mouse IgG-HRP for 1 h. Immunocomplexes on the membranes were visualized with ECL Western Blotting Detection Reagents.

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Real-time PCR analysis of mRNA [1]
HMOX1 expression [1]
Cells grown in plates were treated with statins, Hemin or PGJ2. After 4 h, they were washed twice with ice-cold PBS and collected in the lysis buffer. Total cell RNA was isolated using Perfect Pure RNA Cultured Cell Kit and cDNA was generated using High Capacity RNA-to-cDNA Master Mix according to the manufacturer’s instructions. Real-time PCR for HMOX1 (OMIM *141250) and HMOX2 (OMIM *141251) was performed using the SYBR master mix according to the manufacturer’s instructions with optimized primers. Results were calculated using the comparative Ct method with HPRT as a house-keeping gene and were expressed as % of control.

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Apoptosis evaluation [1]
Apoptosis was quantified using the annexin V-FITC method, which detects phosphatidyl serine externalized in the early phases of apoptosis, in combination with propidium iodide (PI) staining. After exposure to cerivastatin and/or hemin, floating and attached cells were collected, washed with PBS, re-suspended in 100 μl binding buffer and incubated for 20 min at room temperature with 0.3 μl annexin V-FITC. PI (10 μg/ml) was added directly before flow cytometry analysis. Annexin V positive (An+) and PI negative (PI-) are cells in early apoptosis, An + and/or PI positive (PI+) are cells in late apoptosis or post-apoptotic necrosis.

Eight- to ten-week-old male BABL/c mice were used for the ischemia-reperfusion (I/R) experiments. The animals were divided into five groups as follows: (1) the sham group underwent isolation of the bilateral renal arteries without clamping; (2) the vehicle group received an intraperitoneal (i.p.) injection of 4 ml/kg PBS as a vehicle control (with IRI); (3) the Hemin-preconditioned group received Hemin, a potent inducer of HO-1, at 100 μmol/kg i.p.; (4) the Hemin plus ZnPP group received zinc protoporphyrin IX, an inhibitor of HO-1 activity, at 5 mg/kg i.p. 6 h after receiving 100 μmol/kg Hemin i.p.; and (5) the Hemin plus PD98059 group received PD98059, an inhibitor of ERK1/2 activity, at 10 mg/kg i.p. 6 h after receiving 100 μmol/kg Hemin i.p. Both inhibitors were administered i.p. 2 h before I/R, whereas Hemin was administered 8 h before I/R (Supplementary Fig. 1).

[1]. Heme oxygenase is not involved in the anti-proliferative effects of statins on pancreatic cancer cells. BMC Cancer. 2016 May 12;16:309.

[2]. Heme oxygenase-1 ameliorates kidney ischemia-reperfusion injury in mice through extracellular signal-regulated kinase 1/2-enhanced tubular epithelium proliferation. Biochim Biophys Acta. 2015 Oct;1852(10 Pt A):2195-201.

Hemin (trade name Panhematin) is an iron-containing porphyrin. More specifically, it is protoporphyrin IX containing a ferric iron ion (heme B) with a chloride ligand.
Chloro(7,12-diethenyl-3,8,13,17-tetramethyl-21H,23H-porphine-2,18-dipropanoato(4-)-N(21),N(22),N(23),N(24)) ferrate(2-) dihydrogen.

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Drug Indication
Used in the management of porphyria attacks, particularly in acute intermittent porphyria.

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Our data suggest that anti-proliferative effects of statins are not mediated via HMOX pathway. Cerivastatin, the most efficient statin in our study, was capable of inducing several ‘events’ involved in carcinogenesis, including apoptosis, ROS production and inhibition of K-Ras trafficking. Hemin treatment not only substantially decreased cell proliferation independently on HMOX induction, but enhanced anti-proliferative properties of statins in human pancreatic cancer cells. Our findings support the role of statins as agents with potential anti-pancreatic cancer activities. [1]

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The vehicle and hemin-preconditioned groups exhibited the same level of injury to kidney function 24 h after IRI, whereas a markedly decreased level of kidney function was observed 72 h after IRI in the hemin-preconditioned group. These findings might suggest that HO-1 preconditioning can enhance kidney function recovery. In contrast, previous studies found no significant differences in plasma creatinine between HO-1 wild-type and HO-1-deficient mice in the early phase of glycerol-induced AKI. Similarly, our results suggested that the mice that were either subjected to pre-ischemic HO-1 induction or not suffered the same degree of injury in the early phase of AKI. Additionally, Shimizu et al. reported finding no significant changes in serum creatinine in the early phase with or without SnPP treatment in AKI. However, our findings suggested that the renoprotective effect of HO-1 may arise from enhancement of the tubular epithelial cell recovery process.[2]

C34H34CLFEN4O4

653.9562

651.146

C, 62.64; H, 4.95; Cl, 5.44; Fe, 8.57; N, 8.59; O, 9.82

16009-13-5

15953900

Dark purple to black solid powder

300 °C

4.299

2

8

8

43

1010

0

[Fe].Cl[H].O([H])C(C([H])([H])C([H])([H])C1C2=C([H])C3=C(C([H])([H])C([H])([H])C(=O)O[H])C(C([H])([H])[H])=C(C([H])=C4C(C([H])([H])[H])=C(C([H])=C([H])[H])C(C([H])=C5C(C([H])([H])[H])=C(C([H])=C([H])[H])C(=C([H])C(C=1C([H])([H])[H])=N2)N5[H])=N4)[N-]3)=O |c:11,61,t:33,48|

GGIDWJQWCUJYRY-UHFFFAOYSA-L

InChI=1S/C34H34N4O4.Fe/c1-7-21-17(3)25-13-26-19(5)23(9-11-33(39)40)31(37-26)16-32-24(10-12-34(41)42)20(6)28(38-32)15-30-22(8-2)18(4)27(36-30)14-29(21)35-25;/h7-8,13-16H,1-2,9-12H2,3-6H3,(H4,35,36,37,38,39,40,41,42);/q;+3/p-2

3-[18-(2-carboxyethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoic acid;iron(3+)

hemin; 16009-13-5; Chlorohemin; hemin chloride; Haemin; Ferriprotoporphyrin chloride; Protohemin; Chloroprotohemin;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

1M NaOH : 6.67 mg/mL (~10.23 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)