Temozolomide (TMZ)

Biological Activity

Temozolomide (NSC 362856, CCRG 81045) is a DNA methylating agent used for the treatment of Grade IV astrocytoma. Temozolomide exhibits schedule-dependent antineoplastic activity by interfering with DNA replication. Temozolomide displays antitumor activity against a board spectrum of tumors, including leukemias, lymphomas and solid tumors (IC50 = 5.0 μM for cytotoxicity against mouse TLX5 lymphoma cells). Temozolomide induces G2/M arrest and apoptosis through adduction of a methyl group to O6 position of guanine in genomic DNA and functional inactivation of DNA repair protein O(6)-alkylguanine DNA alkyltransferase (AGT) in base excision repair (BER) pathway. Temozolomide treatment triggered ER stress with increased expression of GADD153 and GRP78 proteins, and deceased pro-caspase 12 protein. Temozolomide is currently in a phase II clinical trial in the treatment of melanoma.

Product Citations

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  Sci Adv. 2022 May 13;8(19):eabn1229.

  Translesion DNA synthesis mediates acquired resistance to olaparib plus temozolomide in small cell lung cancer
  Temozolomide (TMZ) purchased from AbMole

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  Biochem Biophys Res Commun. 2021 Sep 10;569:1-9.

  Rapid tumor recurrence in a novel murine GBM surgical model is associated with Akt/PD-L1/vimentin signaling
  Temozolomide (TMZ) purchased from AbMole

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  bioRxiv. 2020 Oct.

  Connexin 43 confers chemoresistance through activating PI3K
  Temozolomide (TMZ) purchased from AbMole

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  Biomedicines. 2020 Jun 4;8(6):151.

  Considering the Experimental use of Temozolomide in Glioblastoma Research
  Temozolomide (TMZ) purchased from AbMole

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  Cancer Lett. 2020 Jun 1;479:1-12.

  Exosome-mediated Transfer of circRNA CircNFIX Enhances Temozolomide Resistance in Glioma
  Temozolomide (TMZ) purchased from AbMole

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  Cell. 2019 Jun 13;177(7):1903-1914.e14.

  Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.
  Temozolomide (TMZ) purchased from AbMole

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  J Clin Transl Sci. 2018 Nov 21;Vol2, Supplement S1, pp.11-12.

  2036 Extracellular matrix as a novel approach to glioma therapy
  Temozolomide (TMZ) purchased from AbMole

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  Cell Death Dis. 2018 Feb 12;9(2):213.

  Autophagy mediates glucose starvationinduced glioblastoma cell quiescence and chemoresistance through coordinating cell metabolism, cell cycle, and survival
  Temozolomide (TMZ) purchased from AbMole

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  Cancer Lett. 2016 Aug 28;379(1):134-42.

  HDAC6 promotes cell proliferation and confers resistance to temozolomide in glioblastoma
  Temozolomide (TMZ) purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Cell Death Dis (2018). Figure 3. Temozolomide (Abmole Bioscience)
	Method	Glucose starvation sensitizes glioblastoma cells to chemotherapies
	Cell Lines	U87 and U251 cells
	Concentrations	200 μM
	Incubation Time	5 d
	Results	Treatment with either temozolomide or carboplatin induced about 50 and 80% GBM cell death under normal and glucose starvation conditions, respectively

	Source	Cell Death Dis (2018). Figure 1. Temozolomide (Abmole Bioscience)
	Method	Glucose starvation sensitizes glioblastoma cells to chemotherapies
	Cell Lines	U87 and U251 cells
	Concentrations	200 μM
	Incubation Time	5 d
	Results	The cytotoxic effect was progressive and by day 5, temozolomide or carboplatin treatment caused 40–60% cell loss in U87 and U251 cells. Glucose starvation nearly doubled the cell loss to 70–90%.

	Source	Cancer letters (2016). Figure 6.Temozolomide was obtained from AbMole (Houston, TX, USA)
	Method	western blot
	Cell Lines	U87 or U251 cells
	Concentrations	200 μM for U87, 50 μM for U251
	Incubation Time	48 h
	Results	TMZ treatment induces the expression of HDAC6 and EGFR (Fig. 6D). Combination use of HDAC6 inhibitors and TMZ abolishes TMZ mediated EGFR and ERK phosphorylation (Fig. 6G).

	Source	Cancer letters (2016). Figure 5.Temozolomide was obtained from AbMole (Houston, TX, USA)
	Method	cell viability assay and cell apotosis(flow cytometry)
	Cell Lines	U87 or U251 or A172 cells
	Concentrations	200, 400 and 800µM or 50, 100 and 200µM or 100, 200 and 400µM
	Incubation Time	48 h
	Results	These data suggest that the combination treatment of HDAC6 inhibitors and TMZ displayed an additive therapeutic effect on glioblastoma.

	Source	Cancer letters (2016). Figure 3.Temozolomide was obtained from AbMole (Houston, TX, USA)
	Method	cell viability assay and cell apotosis(flow cytometry)
	Cell Lines	U87 or U251 cells
	Concentrations	400, 800 and 1200µM or 50, 100 and 200µM
	Incubation Time	48 h
	Results	Our results suggest that the overexpression of HDAC6 in glioblastoma might be an intrinsic mechanism that confers resistance to TMZ.

Protocol (for reference only)

Cell Experiment
Cell lines	A2058, A375, M238 and M249 cell lines
Preparation method	Colony formation assay Depending on the cell line (and plating efficiency), 200–1000 cells were seeded into each well of a 6-well plate and treated as described in detail previously. In the case of primary melanoma cells, 1000 cells were seeded and let grow for 24 days in the presence or absence of drug treatment; because only small colonies formed, the stained colonies were counted under the microscope.Survival of melanoma cells after drug treatment. Five different melanoma cell lines (as indicated), as well as a culture of primary melanoma tissue cells (labeled ‘primary’) were exposed to increasing concentrations of TMZ (diamonds) or NEO212 (circles) for 48 hours, and long-term survival was determined via colony formation assay (CFA).
Concentrations	0, 25, 50, 75, 100µM
Incubation time	48 h

Animal Experiment
Animal models	A375 cells subcutaneous tumor growth model
Formulation	45% glycerol, 45% ethanol, 10% DMSO
Dosages	50 mg/kg
Administration	subcutaneous injection

Chemical Information

Molecular Weight	194.15
Formula	C6H6N6O2
CAS Number	85622-93-1
Solubility (25°C)	DMSO 18 mg/mL
Storage	-20°C, protect from light, sealed

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Bauer M, et al. PLoS One. Human monocytes undergo excessive apoptosis following temozolomide activating the ATM/ATR pathway while dendritic cells and macrophages are resistant.

[2] Lin CJ, et al. PLoS One. Inhibition of mitochondria- and endoplasmic reticulum stress-mediated autophagy augments temozolomide-induced apoptosis in glioma cells.

[3] Lucio Tentori, et al. Blood . Combined treatment with temozolomide and poly(ADP-ribose) polymerase inhibitor enhances survival of mice bearing hematologic malignancy at the central nervous system site

[4] Plowman J, et al. Cancer Res. Preclinical antitumor activity of temozolomide in mice: efficacy against human brain tumor xenografts and synergism with 1,3-bis(2-chloroethyl)-1-nitrosourea.