HIF-1α (hypoxia-inducible factor 1α); HIF-2α; prolyl hydroxylases (PHDs); p53 (no IC50/Ki/EC50 values provided). [2]

In SKOV-33 cells, selenium (100–400 μM; 3 days) promotes inertness [1]. Cellular inertness mediated by caspase-3 is induced by 100–400 μM of selenium methyl ester for three days [1]. Examination [1]

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Se-Methylselenocysteine displayed strong inhibitory effects on cell proliferation and viability of SKOV-3 ovarian cancer cells in dose- and time-dependent manners. [1]
Se-Methylselenocysteine induced apoptosis as evidenced by morphological features of apoptotic nuclei (propidium iodide staining) and DNA fragmentation (sub-G1 accumulation by flow cytometry). [1]
Se-Methylselenocysteine induced caspase-3 activation, shown by a decrease in the 32 kDa procaspase-3 zymogen and increased DEVD-pNa cleavage (1.5-2-fold increase after 3 days exposure to various concentrations). [1]
Se-Methylselenocysteine induced cleavage of PARP (to 85 kDa fragment) and PLC-γ1 (to 65 kDa fragment), downstream targets of activated caspase-3. [1]
Treatment with Se-Methylselenocysteine did not demonstrate cytochrome c accumulation in the cytosol during apoptosis induction (at 100-200 μM for 3 days), unlike sodium selenite. At 400 μM for 3 days, a dramatic increase in cytoplasmic cytochrome c was detected. [1]
Pretreatment with caspase inhibitors (z-VAD-fmk and DEVD-CHO) prevented Se-Methylselenocysteine-induced apoptosis. [1]
Se-Methylselenocysteine treatment resulted in down-regulation of IAP family proteins: human inhibitor of apoptosis protein 1 (HIAP1), X-linked inhibitor of apoptosis protein (XIAP), and survivin. [1]
Se-Methylselenocysteine did not significantly alter expression of Bcl-2 and Bcl-XL proteins. The level of 21 kDa Bax did not change, but an 18 kDa Bax fragment (p18) was detected after treatment. [1]
The 30 kDa subunit of calpain underwent autolysis beginning at 48 h, with significant increase at 72 h after Se-Methylselenocysteine treatment. [1]
Pretreatment with z-VAD-fmk blocked autolysis of calpain subunit and Bax cleavage, while PARP cleavage was not completely blocked. Calpain inhibitor calpeptin (30 μM) inhibited Bax cleavage effectively but did not block PARP cleavage. These results indicate Bax cleavage is mediated by calpain, and calpain activation may be caspase-dependent. [1]

Nude mice given FaDu and A253 head and neck xenografts tumor activity have increased CDDP and cyclophosphamide resistance when treated with selenium selenium (0.2 mg/mouse; face; once daily for 14 days)[2]. For ten months, methyltyrosine (0.75 mg/kg body weight per day) was administered intraperitoneally (IT) to mice suffering from Alzheimer's disease (AD). Reduces oxidative intermediates, neural pathways, and levels of various metal ions. It also inhibits the expression of precursor protein APP and beta-enzyme (BACE1), which lowers the production of amyloid-beta peptide (Aβ), forms fragmented tau hyperphosphorylation, and forms neurofibrillary tangles by promoting protein phosphatase 2A (PP2A) activity (NFT). This protects synaptic proteins and neuronal activity, which in turn improves spatial learning and memory deficits in AD models [3].
Se-Methylselenocysteine (MSC) given orally (0.75 mg per rat per day) for 14 days before and concurrent with chemotherapy significantly protected Fischer rats against lethal doses of cisplatin (9 mg/kg), oxaliplatin (20 mg/kg), and irinotecan (200 mg/kg), reducing or eliminating diarrhoea, stomatitis, and lethality (0% lethality vs 100% with drug alone).
MSC (0.75 mg/rat/day p.o. for 21 days starting 14 days before CTX) reduced CTX (150 mg/kg)-induced diarrhoea from 50% to 0%, stomatitis from 38% to 0%, and lethality from 62% to 25%.
MSC prevented CTX-induced haemorrhagic cystitis in rats: at 24 h post-CTX (150 mg/kg), bladders from MSC-pretreated rats showed only minor oedema with preserved epithelium, compared to severe haemorrhagic cystitis in CTX alone.
MSC protected against CDDP-induced nephrotoxicity: CDDP (6 mg/kg) increased BUN and creatinine levels significantly (P<0.001), whereas MSC + CDDP restored BUN and creatinine to normal levels (P>0.05 vs control). Histologically, CDDP caused dilated tubules with flat epithelium; MSC + CDDP resulted in near-normal kidney histology (89% of animals showed only 3-5% slightly dilated tubules).
MSC protected against CTX-induced alopecia in rats: after shaving, CTX alone caused slow partial hair regrowth over 30-45 days, while MSC + CTX allowed normal hair regrowth comparable to untreated controls.
MSC protected against oxaliplatin-induced myelotoxicity: oxaliplatin (15 mg/kg) caused ~90% myelosuppression on day 8 and ~20-30% on day 24; MSC reduced myelosuppression to ~40-60% on day 8 and completely restored bone marrow histology on day 24.
MSC potentiated antitumour activity: in rats bearing advanced Ward colorectal carcinoma, CDDP (6 mg/kg) alone gave 32% complete response (CR) rate, increased to 75% with MSC; oxaliplatin (10 mg/kg) alone gave 50% CR, increased to 100% with MSC; CDDP lethal dose (9 mg/kg) + MSC gave 75% CR with no toxicity.
In nude mice bearing human FaDu and A253 head and neck xenografts, MSC (0.2 mg/mouse/day p.o. for 14 days) significantly enhanced tumour growth inhibition by CTX (100 mg/kg) and CDDP (8 mg/kg) (P<0.05 vs drug alone).
The antitumour activity of selenium in combination with chemotherapy is selenium-type dependent; MSC showed superior activity compared to selenomethionine, sodium selenite, and selenised yeast. [2]

MDA (malondialdehyde) content assay: Brain homogenates were measured using a commercial MDA kit according to the manual. MDA, a final product of lipid oxidation, was used as a marker of oxidative stress. [3]
GPx (glutathione peroxidase) activity assay: Brain homogenates were measured using a Glutathione Peroxidase Detection Kit following the manual. GPx reduces lipid hydroperoxides or hydrogen peroxide to corresponding alcohols or water. [3]
SOD (superoxide dismutase) activity assay: Brain homogenates were measured using a Total SOD Activity Detection Kit according to the manual. SOD catalyzes the dismutation of superoxide radical into oxygen and hydrogen peroxide. [3]
ICP-MS (Inductively Coupled Plasma Mass Spectrometry) for metal quantification: Brain homogenate (100 μl in lysis buffer) was digested with pure electronic nitric acid (400 μl) in a microwave digestion system at 45°C for 16 h. Digested liquid was diluted with 1% nitric acid to 5 mL. Concentrations of Cu, Fe, Zn, Ba, V, Cr, Co and Mn were measured using ICP-MS under conditions: forward power 1300 W, cool gas flow 13.5 L/min, auxiliary gas flow 1.1 L/min, nebuliser gas flow 0.89 L/min. [3]
AFS (Atomic Fluorescence Spectrometry) for Se and Hg: Concentrations were measured using an AFS system with conditions: PTM voltage 270 V, atomizer height 8 mm, lamp current 30 mA (Hg) or 80 mA (Se), carrier gas flow 400 ml/min, shield gas flow 800 ml/min, curve read time 9 s, delay time 0.5 s. [3]
SR-μXRF (Synchrotron Radiation X-ray Fluorescence) for metal distribution: Brain sections (50 μm thick) were analyzed at beamline BL15U of SSRF. Photo energy 12.95 keV, focused beam 100×100 μm² with 2 s irradiation per spot. Fluorescence counts of Cu, Fe and Zn were extracted using GeoPIXE software, and intensity maps were generated with Plot2D. [3]
Western blot analysis: Brain tissues were dissolved in lysis buffer with PMSF and protease inhibitor cocktail. Proteins separated by SDS-PAGE, transferred to PVDF membranes, probed with primary antibodies, then HRP-conjugated secondary antibodies. Bands visualized with ECL detection reagents and quantified with Quantity One software. [3]
Immunofluorescence for Aβ: Brain sections (5 μm) treated with citrate buffer (0.01 mol/L, pH 6.0) at 4°C for 5 min, blocked with 10% goat serum in PBS, incubated with primary antibodies and Dylight-488 or Dylight-594 conjugated secondary antibodies. Images captured by confocal microscope and analyzed with Image Pro Plus. [3]
Gallyas-Braak silver staining for NFTs: Performed as described previously to analyze NFTs in brain sections. Images collected on Olympus BX51 microscope with 40× objective. NFT positive areas evaluated using Image Pro Plus. [3]
Nissl staining: Brain sections stained with 0.5% cresyl violet for 10 min after washing twice with PBS, dehydrated in graded ethanol, placed in xylene, mounted. Nissl bodies visualized by confocal microscope and positive cells assessed by Image Pro Plus. [3]

Apoptosis Analysis[1]
Cell Types: SKOV-3 Cell
Tested Concentrations: 100, 200, 400 μM
Incubation Duration: 3 days
Experimental Results: Resulted in a significant increase in the accumulation of Sub-G1 phase, which occurred in a SeMSC concentration and culture time dependence.

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Western Blot Analysis[1]
Cell Types: SKOV-3 Cell
Tested Concentrations: 100, 200, 400 μM
Incubation Duration: 3 days
Experimental Results: Resulted in diminished expression of the 32 kDa form of procaspase-3.

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SKOV-3 ovarian cancer cells were maintained in RPMI1640 medium with 10% fetal bovine serum, 2 mM glutamine, and antibiotics at 37°C in 5% CO2. Cells were seeded at 10^5/ml in T-25 flasks, cultured for 18-20 h before Se-Methylselenocysteine addition. Cell number and viability were determined using hemocytometer and trypan blue exclusion. [1]
Assessment of apoptotic nuclei by propidium iodide (PI) staining: Cells were harvested, washed with PBS, fixed with 4% paraformaldehyde in 0.1% phosphate buffer for 10 min at room temperature, followed by ethanol containing 1% HCl for 10 min at -20°C. Fixed cells were placed on slides and stained with 1 μg/ml PI solution containing 100 μg/ml DNase-free RNase A for 30 min at 37°C. Nuclear morphology examined by fluorescence microscopy. [1]
Flow cytometry analysis for sub-G1 DNA: Cells were harvested, washed with PBS, fixed in ice-cold 70% ethanol and stored at 4°C. Prior to analysis, cells were washed with PBS, suspended in 1 ml of cold PI solution (0.5 mg/ml RNase, 50 μg/ml propidium iodide, 0.1% sodium citrate, 0.1% NP-40), incubated on ice for 30 min in the dark. Cytometric analyses were performed using flow cytometer; approximately 15,000 cells counted per determination. [1]
Caspase-3 like activity measurement: Cells were collected and washed with PBS, resuspended in 25 mM HEPES (pH 7.5), 5 mM MgCl2, 5 mM EDTA, 5 mM DTT, 2 mM PMSF, and 1 μg/ml each of aprotinin, leupeptin, pepstatin. Cell lysates clarified by centrifugation at 12,000 rpm for 10 min. Clear lysates containing 50 μg protein were incubated with 50 μM Ac-DEVD-pNa colorimetric substrate. Optical density at 405 nm was measured after incubation at 37°C for 1 h. [1]
Western blot analysis: Whole cell extracts prepared in lysis buffer. Mitochondria-free cell extracts prepared. Protein concentration estimated with Bradford reagent. 50-100 μg protein per sample separated on 8-12% SDS-PAGE, transferred to Immobilon P membranes. Immunoblotting performed using alkaline phosphatase-conjugated secondary antibody. Equal protein loading confirmed by Ponceau Red staining. [1]

Animal/Disease Models: Female athymic nude mice (bearing human A253 and FaDu squamous cell carcinoma xenografts) [ 2]
Doses: 0.2mg/only
Route of Administration: po; ]. one time/day for 14 days (7 days before and 7 days after cyclophosphamide or CDDP, 14 days total) Experimental Results:

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Fischer rats (12-24 per group, 3-6 experiments): Se-Methylselenocysteine (MSC) was administered orally (p.o.) daily at 0.75 mg per rat. For protection studies, MSC was given for 14 days before chemotherapy and continued daily during and after treatment (total up to 21-35 days). Cyclophosphamide (CTX), cisplatin (CDDP), and oxaliplatin were given as a single intravenous (i.v.) injection; irinotecan was given i.v. weekly ×4. MTD of MSC was 0.75 mg/rat/day for up to 42 days; a single dose of 1.5 mg/rat was highly toxic. Optimal protection was achieved only when MSC treatment preceded drug treatment. [2]
Alopecia evaluation in rats: areas on back (~7×9 cm) were shaved; animals were untreated control, CTX alone (100 mg/kg i.v. ×1), or CTX + MSC (0.75 mg/rat/day p.o. daily for 21 days starting 14 days before CTX). Hair regrowth evaluated on days 0, 30, 45 after CTX, followed for 90 days. 4 rats per group, treatments repeated three times. [2]
Genitourinary toxicity evaluation in rats: bladders removed at 4 h and 24 h post-CTX (100 or 150 mg/kg). Kidneys removed on day 6 after CDDP (6 mg/kg). Blood samples collected by cardiac puncture for BUN and serum creatinine测定 using commercial kits (urease method and creatinine amidohydrolase method). Tissues fixed in 10% neutral buffered formalin, paraffin-embedded, 5 μm sections stained with haematoxylin-eosin for histopathology. Groups: untreated control, MSC alone (0.75 mg/rat/day), CTX or CDDP alone, and CTX or CDDP + MSC. 9 rats per group from two independent experiments. [2]
Tumour measurement and antitumour activity in rats: Ward colorectal carcinoma transplanted; treatments started when tumours reached ~2500-3000 mg (14 days after transplantation). MSC 0.75 mg/rat/day p.o. started 14 days before chemotherapy. Tumour weight calculated as ½(L×W²). Partial response (PR): ≥50% tumour reduction; complete response (CR): tumour undetectable; cure: survival with no tumour at 60 days. For oxaliplatin alone (10 mg/kg) group, 12 rats; other groups 4 rats. [2]
Nude mice bearing FaDu and A253 xenografts: tumours reached ~200-250 mg (7 days after transplantation). MSC 0.2 mg/mouse/day p.o. for 7 days before and 7 days after CTX (100 mg/kg i.v. ×1) or CDDP (8 mg/kg i.v. ×1), total 14 days. 5 mice per group. [2]
Bone marrow toxicity study in rats: oxaliplatin at MTD (15 mg/kg i.v. ×1) with or without MSC (0.75 mg/rat/day p.o. daily for 21 days starting 14 days before oxaliplatin). Sternums removed on days 8 and 24, decalcified, sectioned, stained with haematoxylin-eosin. [2]

Se-Methylselenocysteine is orally bioavailable.
The desired plasma concentration of selenium for optimal modulation of therapeutic efficacy and selectivity in preclinical models is 20-30 μM.
Therapeutic concentrations can be achieved in vivo without toxicity.
MSC requires one-step activation by β-lyase and has relatively less serum protein binding/interaction compared to other selenium forms. [2]

Maximum tolerated dose (MTD) of Se-Methylselenocysteine (MSC) in normal and tumour-bearing Fischer rats was 0.75 mg per rat per day (administered orally for up to 42 days). A single oral dose of 1.5 mg per rat was highly toxic.
MSC alone at 0.75 mg/rat/day caused no weight loss, diarrhoea, stomatitis, or lethality in rats (mean weight loss 5.8 ± 1.2%, all toxicity parameters 0%).
In nude mice, MSC at 0.2 mg/mouse/day p.o. for 14 days had no significant antitumour activity but was well tolerated.
MSC at 0.75 mg/rat/day prevented or reduced chemotherapy-induced toxicities including diarrhoea, stomatitis, alopecia, bladder haemorrhagic cystitis, nephrotoxicity, and myelosuppression without causing additional toxicity. [2]

[1]. Se-methylselenocysteine induces apoptosis through caspase activation and Bax cleavage mediated by calpain in SKOV-3 ovarian cancer cells. Cancer Lett. 2002 Aug 8;182(1):83-92.

[2]. Se-methylselenocysteine offers selective protection against toxicity and potentiates the antitumour activity of anticancer drugs in preclinical animal models. Br J Cancer. 2014 Apr 2;110(7):1733-43.

[3]. Se-Methylselenocysteine Ameliorates Neuropathology and Cognitive Deficits by Attenuating Oxidative Stress and Metal Dyshomeostasis in Alzheimer Model Mice. Mol Nutr Food Res. 2018 Jun;62(12):e1800107.

Selenomethyl-L-selenocysteine is an L-α-amino acid compound with a methylselenomethyl side chain. It possesses antitumor activity. Selenomethyl-L-selenocysteine is a non-protein L-α-amino acid and a derivative of L-selenocysteine. It is the conjugate base of selenomethyl-L-selenocysteine salt, the conjugate acid of selenomethyl-L-selenocysteine acid, and the enantiomer of selenomethyl-D-selenocysteine. It is a zwitterion tautomer of selenomethyl-L-selenocysteine. Methylselenocysteine has been used in clinical trials for the prevention of prostate cancer and in which there is no evidence of disease. Selenomethylselenocysteine is a metabolite found or produced in Escherichia coli (K12 strain, MG1655 strain). It has been reported that selenomethylselenocysteine is present in cattle, Astragalus membranaceus, and Eupolyphaga sinensis, and relevant data are available. Methylselenocysteine is a naturally occurring organoselenium compound found in many plants, including garlic, onions, and broccoli, and possesses potential antioxidant and chemopreventive activities. Selenomethylselenocysteine (MSC) is an amino acid analog of cysteine in which the methyl selenium atom replaces the sulfur atom in cysteine. This substance exerts its antioxidant effect upon binding to glutathione peroxidase and has shown potent chemopreventive activity in animal models.

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Se-Methylselenocysteine is a chemopreventive agent that induces apoptosis in SKOV-3 ovarian cancer cells through a cytochrome c-independent caspase-3 activation, down-regulation of IAP family proteins (HIAP1, XIAP, survivin), and Bax cleavage mediated by caspase-dependent calpain activation. The chemopreventive effects of Se-MSC may be related to caspase-3 activation, down-regulation of IAP family proteins, and Bax cleavage. [1]

C4H9NO2SE

182.081

182.979

26046-90-2

Se-Methylselenocysteine hydrochloride;863394-07-4

147004

White to off-white solid powder

314.1±37.0 °C at 760 mmHg

177 °C(dec.)

143.7±26.5 °C

0.0±1.4 mmHg at 25°C

-0.89

2

3

3

8

86.1

1

C[Se]C[C@@H](C(=O)O)N

XDSSPSLGNGIIHP-VKHMYHEASA-N

InChI=1S/C4H9NO2Se/c1-8-2-3(5)4(6)7/h3H,2,5H2,1H3,(H,6,7)/t3-/m0/s1

(2R)-2-amino-3-methylselanylpropanoic acid

Se-Methylselenocysteine Se-MSC MSeCSeMCys SeMSCSe MSC

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

H2O : ~83.33 mg/mL (~457.66 mM)
DMSO :< 1 mg/mL

Solubility in Formulation 1: 50 mg/mL (274.60 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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