homologous nucleoside antibiotic; N-linked glycosylation; GlcNAc phosphotransferase (GPT)
DPAGT1 (UDP-GlcNAc-dolichol-phosphate N-acetylglucosamine-1 phosphate transferase) [2]

GRP78 (glucose-regulated protein-78) – upregulation of GRP78 expression contributes to resistance [1]

Skp2 – downregulation of Skp2 leads to p27 accumulation [3]

In CD44+/CD24- and primary MCF7 cells, tunitamycin (2 μg/mL; 24 hours) treatment enhanced fragment-associated XBP-1, ATF6 nuclear translocation levels, and CHOP protein expression [1]. The findings demonstrate that CD44+/CD24- and CD44+/CD24-rich MCF7 cell cultures exhibit the following effects of tunicamycin: they decrease migration, increase cell death, and block migration [1].

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In Hep3B human hepatocellular carcinoma cells, tunicamycin (1 μg/ml) significantly inhibited apoptosis induced by topoisomerase inhibitors camptothecin (3 μM) and etoposide (5 μM), but not by tubulin-targeting drugs taxol (0.1 μM) and vincristine (0.1 μM). Tunicamycin increased G1 phase population (G1 arrest) and reduced sub-G1 population. Tunicamycin did not modify topoisomerase levels (TOP I, TOP IIα, IIβ) nor inhibit ATM activation caused by camptothecin and etoposide. Tunicamycin blocked camptothecin- and etoposide-induced cleavage of Bcl-2 family proteins (Bid, Bad, Bax) and activation of caspase-3 and caspase-7. Tunicamycin also blocked camptothecin-induced upregulation of cyclin E, cyclin A, and Cdk2, and etoposide-induced increase of cyclin A and Cdk2. Tunicamycin alone induced time-dependent G1 arrest. Post-addition experiments showed that the resistance effect decreased when tunicamycin was added later after topoisomerase inhibitor treatment. Curcumin (30 μM), another G1 arrest inducer, similarly inhibited camptothecin- and etoposide-induced apoptosis. GRP78 knockdown by siRNA (sequence: 5'-AUA ACA UUU AGG CCA GCA AUA GUU C-3') partially reversed tunicamycin-induced resistance to camptothecin but did not affect tunicamycin-induced inhibition of cell-cycle regulators, indicating GRP78 and G1 arrest are independent factors [1].

In multiple hepatocellular carcinoma cell lines (MHCC-97L, MHCC-97H, Huh7, SMMC-7721, PLC/PRF/5, HCC-LY5), tunicamycin (2.5 μg/ml for 24 h) dramatically reduced ABCG2 protein expression in a time- and dose-dependent manner, and inhibited Akt phosphorylation. Tunicamycin (2.5 μg/ml for 24 h) altered ABCG2 subcellular localization from plasma membrane to intracellular aggregation. Tunicamycin reduced the side population (SP) rate related to ABCG2 efflux activity. Tunicamycin (1.0 μg/ml for 48 h) reduced expression of CSC markers (CD133, CD44, CD13, EpCAM, CK-19) and decreased PCNA expression while increasing cleaved PARP. Combination of tunicamycin (1.0 μg/ml) with 5-FU (100 μg/ml in MHCC-97L; 10 μg/ml in Huh7) or cisplatin (20 μg/ml in MHCC-97L; 3.5 μg/ml in Huh7) for 48 h further reduced ABCG2, p-Akt, PCNA, and CSC markers, and enhanced cleaved PARP compared to single agents. Overexpression of ABCG2 or constitutively active Akt (Akt-myr) partially rescued the sensitization effect of tunicamycin to cisplatin [2].

In melanoma cell lines (Mel-RM, A375, MEWO, MM200, Mel4405, IGR3), tunicamycin (3 μM) induced time-dependent G1 cell cycle arrest, increased p27 protein levels without affecting cyclin D1 levels. p27 knockdown by shRNA rescued the G1 arrest. Tunicamycin prolonged the half-life of p27 (from ~4 h to >8 h) by inhibiting its ubiquitin-dependent proteasomal degradation. Tunicamycin reduced Skp2 protein levels in a time-dependent manner, but not Pirh2. The decrease in Skp2 was at the transcriptional level (mRNA downregulation) as shown by real-time PCR. In vitro ubiquitination assay using GST-p27 incubated with lysates from tunicamycin-treated cells showed reduced polyubiquitination of p27 [3].

In a CD133+/- MHCC97L cell xenograft model (BALB/c (nu/nu) mice), tunitoxin (0.1 mg/kg or 0.5 mg/kg) therapy greatly reduces tumor growth [2].

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In BALB/c (nu/nu) mouse xenograft models using CD133+ MHCC-97L cells, treatment with tunicamycin (0.5 mg/kg) for 4 weeks resulted in fewer (3 of 6 mice) and smaller tumors compared to control group. Tunicamycin decreased ABCG2 and p-Akt protein levels in xenograft tumors [2].

In MHCC-97L and Huh7 xenograft nude mouse models, combination therapy of tunicamycin (dose not explicitly specified in main text, but likely 0.5 mg/kg) with low-dose cisplatin dramatically suppressed tumor growth compared to single-agent treatments, as measured by tumor weight and volume [2].

To assess N-linked glycosylation of ABCG2, cell lysates from MHCC-97L and MHCC-97H were treated with PNGase F, Endo Hf, or α-2,3-neuraminidase at 37°C according to standard protocols. The shift in ABCG2 molecular weight was detected by Western blotting, confirming that ABCG2 is glycosylated [2].

For in vitro ubiquitination assay of p27, GST-p27 fusion protein expressed in E. coli was purified and immobilized on glutathione sepharose beads. The beads-bound GST-p27 was then incubated with lysates from Mel-RM cells treated with or without tunicamycin (3 μM for 30 h, followed by 6 h MG132) for 3 hours. After incubation, beads were washed and eluted proteins were subjected to Western blot analysis with anti-ubiquitin antibody to detect polyubiquitinated forms of p27 [3].

Western Blot Analysis
Cell Types: CD44+/CD24- and Original MCF7 Cells[1]
Tested Concentrations: 2 µg/mL
Incubation Duration: 24 hrs (hours)
Experimental Results: Spliced XBP-1, ATF6 nuclear transfer detected in CD44+/CD24- and original MCF7 cells and CHOP protein expression levels increased.

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Hepatocellular carcinoma is chemoresistant to many anticancer drugs. Tunicamycin, an N-glycosylation inhibitor, causes unfolded protein response and is widely used as pharmacological inducer of endoplasmic reticulum stress. In this study, several designs were used to investigate the resistance mechanism to camptothecin and etoposide in hepatocellular carcinoma Hep3B cells. Tunicamycin significantly inhibited apoptosis induced by camptothecin or etoposide. Tunicamycin neither modified the topoisomerase levels nor inhibited the ATM activation caused by camptothecin and etoposide. The data suggest that tunicamycin-induced resistance may result from the downstream events of drug-trapped topoisomerase-DNA complexes and DNA double-strand breaks. Camptothecin and etoposide caused an increase of protein expression of several cell-cycle regulators and induced the cleavage of Bcl-2 family of proteins. These intracellular molecular events were abolished by tunicamycin. A design of postaddition of tunicamycin demonstrated that G1 checkpoint arrest contributed to the resistance mechanism. Curcumin, another G1 arrest-inducing agent in this study, was able to induce a similar resistant effect. Furthermore, the cells transfected with GRP78 siRNA were partly resistant to tunicamycin-induced apoptosis but not the inhibitory effect on cell-cycle regulators indicating that GRP78 and G1 arrest are two independent factors to tunicamycin-induced resistance mechanism. In conclusion, the data suggest that tunicamycin induces the resistance to topoisomerase inhibitors through GRP78 up-regulation and G1 arrest of the cell cycle. The findings also prompt the deliberation that the resistance can be caused during combined administration of chemotherapeutic drugs and Chinese herbal medicines, which induce endoplasmic reticulum stress and/or cell-cycle arrest in cancer cells.[1]

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Hep3B cells were cultured in RPMI-1640 medium with 10% FBS and penicillin/streptomycin. After treatment with vehicle (0.1% DMSO) or agents (tunicamycin 1 μg/ml; camptothecin 3 μM; etoposide 5 μM; taxol 0.1 μM; vincristine 0.1 μM; curcumin 30 μM) for indicated times, cells were harvested, fixed with 70% alcohol at 4°C for 30 min, washed with PBS, incubated in phosphate-citric acid buffer for 30 min at room temperature, then resuspended in propidium iodide solution (containing Triton X-100, RNase, and PI). DNA content was analyzed by FACScan flow cytometry and CellQuest software. For Western blotting, cells were lysed in ice-cold lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, aprotinin, leupeptin, 1% Triton X-100). Proteins (40 μg) were separated by 10% or 15% PAGE, transferred to nitrocellulose membrane, blocked with PBS/5% nonfat milk, and immunoblotted with primary antibodies (against GRP78, TOP I, TOP II, Bcl-2, Bad, Bid, Bax, cyclins, Cdks, phospho-ATM, caspases, etc.), followed by HRP-conjugated secondary antibodies and chemiluminescence detection. For siRNA transfection, cells at 50% confluence were transfected with GRP78 siRNA (5'-AUA ACA UUU AGG CCA GCA AUA GUU C-3') or control siRNA using Lipofectamine 2000 for 6 h, then treated with agents [1].

Hepatocellular carcinoma cell lines (Huh7, PLC/PRF/5, SMMC-7721, MHCC-97L, MHCC-97H, MHCC-LM3, HCC-LY5) were cultured in DMEM with 10% FBS. For cell sorting, PLC/PRF/5 cells were labeled with PE-conjugated anti-human CD133/1 antibody and sorted by FACS. Other cell lines with <1% CD133+ cells were magnetically isolated using EasySep PE Selection Kit. For real-time PCR, total RNA was extracted with TRIzol, reverse transcribed, and PCR performed with SYBR Premix Ex Taq using specific primers (e.g., for ABCG2, GAPDH). For Western blotting, cells were lysed and proteins detected with antibodies against ABCG2, Akt, p-Akt, PCNA, cleaved PARP, CD133, CD44, etc. For immunostaining, cells on culture slides were treated with tunicamycin (2.5 μg/ml) or LY294002 (10 mmol/L) for 24 h, then fixed and stained with BXP-21 anti-ABCG2 antibody and DAPI, and observed by confocal microscopy. For SP analysis, cells were stained with Hoechst 33342 and analyzed by flow cytometry. For lentiviral transfection, ABCG2 cDNA or Akt-myr was cloned into pWPXL, virus packaged in HEK 293T cells, and target cells transduced [2].

Melanoma cells (Mel-RM, A375, MEWO, MM200, Mel4405, IGR3) were cultured as described. For cell cycle analysis, cells were harvested, fixed with 70% ethanol at 4°C overnight, washed, and incubated in PBS containing propidium iodide (40 mg/ml) and RNase A (200 mg/ml) for 30 min at room temperature, then analyzed by flow cytometry. For Western blotting, cell lysates were probed with anti-p27, anti-cyclin D1, anti-GRP78, anti-Skp2, anti-Pirh2, anti-GAPDH antibodies. For semi-quantitative RT-PCR, total RNA was reverse transcribed and amplified with primers for p27 and Skp2. Real-time PCR for Skp2 used SYBR green with actin as internal control. For ubiquitination assay, cells transfected with HA-ubiquitin were treated with tunicamycin (3 μM) for 30 h followed by MG132 for 6 h, then lysed and immunoprecipitated with anti-p27 antibody; immunoprecipitates were immunoblotted with anti-ubiquitin and anti-p27 antibodies. For half-life determination, cells were pretreated with DMSO, MG132, or tunicamycin for 28 h, then incubated with cycloheximide (CHX) for indicated times, and p27 levels analyzed by Western blotting [3].

Six- to 8-week-old BALB/c (nu/nu) mice were maintained under standard conditions according to protocols developed by the Shanghai Medical Experimental Animal Care Commission. At the end of the experimental period, the mice were euthanized, and the excised tumors were weighed and fixed in formalin. Frozen tumor samples were analyzed by Western blotting and real-time PCR. Details can be found in the Supplementary Data.[2]

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Six- to eight-week-old BALB/c (nu/nu) mice were maintained under standard conditions. For xenograft assay, MHCC-97L or Huh7 cells were injected subcutaneously (details in Supplementary Data). For evaluating tunicamycin effect on CD133+ CSCs, CD133+ and CD133- MHCC-97L cells were implanted into nude mice. After tumor establishment, mice were treated with tunicamycin (0.5 mg/kg) for 4 weeks (route not specified, likely intraperitoneal). At the end of the experiment, mice were euthanized, tumors excised, weighed, and fixed in formalin. Frozen tumor samples were analyzed by Western blotting and real-time PCR for ABCG2 and p-Akt expression [2].

[1]. Tunicamycin induces resistance to camptothecin and etoposide in human hepatocellular carcinoma cells: role of cell-cycle arrest and GRP78. Naunyn Schmiedebergs Arch Pharmacol. 2009 Nov;380(5):373-82.

[2]. DPAGT1/Akt/ABCG2 pathway in mouse Xenograft models of human hepatocellular carcinoma. Mol Cancer Ther. 2013 Dec;12(12):2874-84.

[3]. Endoplasmic reticulum stress inhibits cell cycle progression via induction of p27 in melanoma cells. Cell Signal. 2013 Jan;25(1):144-9.

[4]. Mechanisms associated with biogenesis of exosomes in cancer. Mol Cancer. 2019 Mar 30;18(1):52.

Hepatocellular carcinoma (HCC) exhibits high chemoresistance, and ATP-binding cassette transporter G subfamily member 2 (ABCG2) is considered to play a key role in this resistance. This study aimed to develop effective therapeutic strategies to reduce ABCG2 expression levels and overcome chemoresistance in HCC. First, we demonstrated a positive correlation between ABCG2 protein levels and chemoresistance in HCC cancer cell lines. ABCG2 is preferentially expressed in CD133-rich, highly resistant HCC cancer stem cells (CSCs). Furthermore, ABCG2 undergoes N-glycosylation modification in HCC cells, a modification involved in maintaining its protein stability. The N-glycosylation (NLG) inhibitor tunicamycin significantly reduced ABCG2 expression, altered its subcellular localization, and reversed its drug efflux effect in various HCC cancer cell lines. In addition, tunicamycin reduced the expression levels of several cancer stem cell (CSC) markers and inhibited the tumorigenicity of CD133(+) CSCs. Chlamycin combined with cisplatin (CDDP) can inhibit the expression of proliferating cell nuclear antigen (PCNA) and increase PARP cleavage; overexpression of ABCG2 or Akt-myr can partially reverse this effect. Compared with monotherapy, combination therapy can more effectively inhibit tumor growth in xenograft mice. Finally, CDDP treatment combined with UDP-GlcNAc-polyterpenol-phosphoglucosamine-1-phosphotransferase (DPAGT1) knockdown can reproduce the effect observed when CDDP is used in combination with chlamycin. In summary, our results suggest that chlamycin may reverse drug resistance in hepatocellular carcinoma by targeting the DPAGT1/Akt/ABCG2 pathway and improve the efficacy of combination therapy [2].

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The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a stress signaling pathway. UPR coordinates the induction of ER molecular chaperones, the reduction of protein synthesis, and growth arrest in the G1 phase of the cell cycle. However, the molecular mechanism by which UPR induces G1 phase cell cycle arrest remains unclear. This article reports that tunicamycin (TM, an ER stress inducer) activates the UPR response, leading to the accumulation of p27 and G1 phase cell cycle arrest in melanoma cells. The accumulation of p27 is due to the inhibition of its polyubiquitination and subsequent degradation by TM treatment. Associated with p27 stabilization is the reduced level of the p27 E3 ligase Skp2 after TM treatment. More importantly, knockdown of p27 significantly reduces TM-induced G1 phase cell cycle arrest. In summary, these data suggest that p27 is a key mediator of endoplasmic reticulum stress-induced growth arrest. [3]

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Tunicamycin is an N-glycosylation inhibitor that induces endoplasmic reticulum stress and the unfolded protein response. It upregulates GRP78 (an ER chaperone) and causes G1 cell cycle arrest. In hepatocellular carcinoma, tunicamycin can induce resistance to topoisomerase inhibitors (camptothecin, etoposide) via GRP78 upregulation and G1 arrest, suggesting caution when combining ER stress-inducing Chinese herbal medicines with chemotherapy [1]. In contrast, tunicamycin potentiates cisplatin efficacy in hepatocellular carcinoma by downregulating ABCG2 and suppressing cancer stem cells via the DPAGT1/Akt/ABCG2 pathway, offering a potential combination strategy [2]. In melanoma cells, tunicamycin-induced ER stress leads to G1 arrest through p27 accumulation via transcriptional downregulation of Skp2 [3].

C₃₉H₆₄N₄O₁₆(N=₁₀)

844.94 (n=10)

844.94 (n=10)

11089-65-9

Off-white to light yellow solid powder

1.4±0.1 g/cm3

234 - 235ºC

1.617

2.58

ZHSGGJXRNHWHRS-VIDYELAYSA-N

InChI=1S/C30H46N4O16/c1-11(2)5-4-6-16(38)32-19-23(43)20(40)14(47-29(19)50-28-18(31-12(3)36)22(42)21(41)15(10-35)48-28)9-13(37)26-24(44)25(45)27(49-26)34-8-7-17(39)33-30(34)46/h4,6-8,11,13-15,18-29,35,37,40-45H,5,9-10H2,1-3H3,(H,31,36)(H,32,38)(H,33,39,46)/b6-4+/t13?,14-,15-,18-,19-,20+,21-,22-,23-,24+,25-,26-,27-,28-,29+/m1/s1

(E)-N-((2S,3R,4R,5R,6R)-2-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6-(2-((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)-2-hydroxyethyl)-4,5-dihydroxytetrahydro-2H-pyran-3-yl)-5-methylhex-2-enamide

NSC 177382; NSC177382; NSC-177382

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)

DMSO : ~33.33 mg/mL
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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