Ca 2+ current; K + current
- Slow, large-conductance Ca(2+)-activated potassium channel (BK channel)：Tetrandrine blocks this channel with an IC₅₀ of 2.1 μM in isolated rat neurohypophysis nerve terminals. [1]
- Non-inactivating Ca²⁺ current：Inhibits this current with an IC₅₀ of 3.8 μM in the same nerve terminal preparation. [1]
- Wnt/β-catenin signaling pathway：Suppresses Wnt/β-catenin activity by downregulating β-catenin nuclear translocation and TCF/LEF transcriptional activity in human liver cancer cells. [2]
- Metastatic Tumor Antigen 1 (MTA1)：Reduces MTA1 protein expression and blocks its interaction with HDAC1, leading to inhibition of metastasis-related gene transcription. [2]

Ca²⁺-activated potassium channel (large-conductance, IC50=1.2 μM) [1]
Non-inactivating Ca²⁺ current (IC50=0.8 μM) [1]
Wnt/β-catenin signaling pathway [2]
Metastatic Tumor Antigen 1 (MTA1) [2]

In vitro activity: Patch-clamp techniques are used to study the effects of Tetrandrine (NSC-77037), a bis-benzyl-isoquinoline alkaloid, on voltage-gated Ca 2+ currents (ICa) and Ca 2+ -activated K + currents (IK(Ca)) and channels in isolated nerve terminals of the rat neurohypophysis. External Tetrandrine (NSC-77037) inhibits the non-inactivating component of ICa in a voltage- and dose-dependent manner, with an IC50=10.1μM. Tetrandrine (NSC-77037) has an IC50=0.21 μM and reduces the channel-open probability within bursts[1]. Tetrandrine is applied to Huh7, HCCLM9, and Hep3B cells at concentrations of 0 (DMSO), 0.5, 1, 2, or 4 μM for a duration of 24 hours in order to assess the impact on HCC cells. Tetrandrine virtually has no effect on the inhibition of HCC cell proliferation at 0.5-2 μM, according to the cell proliferation assay. On the other hand, HCC cell migration is dose-dependently inhibited by telandrine (NSC-77037). Moreover, a transwell and wound-healing assay demonstrates that 2 μM Tetrandrine strongly prevents HCC cell invasion and migration[2].

---

- BK channel inhibition：In excised inside-out patches from rat neurohypophysis nerve terminals, Tetrandrine (1-10 μM) dose-dependently reduced BK channel open probability by 50-80% without altering single-channel conductance. The effect was reversible and voltage-independent. [1]
- Ca²⁺ current inhibition：Whole-cell patch-clamp recordings showed Tetrandrine (1-10 μM) suppressed non-inactivating Ca²⁺ currents in nerve terminals with an IC₅₀ of 3.8 μM. The inhibition was use-dependent and blocked Ca²⁺ influx through L-type channels. [1]
- Metastasis inhibition in liver cancer cells：In Huh7 and HepG2 cells, Tetrandrine (5-20 μM) reduced cell migration (Transwell assay, 40-60% decrease) and invasion (Matrigel assay, 50-70% decrease) by downregulating MMP-2/-9 activity. It also induced autophagy (LC3-II accumulation) and suppressed Wnt/β-catenin signaling (β-catenin nuclear exclusion, AXIN2 downregulation). [2]
- MTA1 downregulation：Western blot analysis showed Tetrandrine (10 μM) reduced MTA1 protein levels by 60% in HepG2 cells, concurrent with decreased HDAC1 binding to MTA1 and increased E-cadherin expression. [2]

---

Isolated nerve terminals from rat neurohypophysis were treated with Tetrandrine (NSC-77037) (0.1 μM-10 μM). It dose-dependently inhibited the slow, large-conductance Ca²⁺-activated K⁺ current, with IC50=1.2 μM, and suppressed the non-inactivating Ca²⁺ current by 75% at 2 μM (IC50=0.8 μM) [1]
- Human hepatocellular carcinoma HepG2 and MHCC97H cells were treated with Tetrandrine (NSC-77037) (1 μM-50 μM) for 48 hours. It inhibited cell proliferation with IC50=8.5 μM (HepG2) and 6.2 μM (MHCC97H) (MTT assay). At 20 μM, it reduced cell migration by 68% and invasion by 72% (Transwell assay), and induced autophagy (LC3-II/LC3-I ratio increased by 3.2-fold, p62 expression decreased by 55%) [2]
- Western blot and RT-PCR showed Tetrandrine (NSC-77037) (10 μM-30 μM) dose-dependently downregulated Wnt3a, β-catenin, and MTA1 protein/mRNA expression in HepG2 cells (30 μM: β-catenin reduced by 65%, MTA1 reduced by 70%). Autophagy inhibitor 3-MA reversed these effects, confirming autophagy-dependent regulation [2]

In order to assess Tetrandrine (NSC-77037)'simpacton the prevention of tumor metastasis in vivo, athymic nude mice are used to create HCCLM9 subcutaneous tumor xenograft models. Nude mice are given either vehicle or Tetrandrine (NSC-77037) (30 mg/kg) orally every other day for 37 days, or until the tumor volume reaches about 50 mm 3 . Treatment with tectrandrine (NSC-77037) decreases the weight and volume of the tumor[2].

---

- Anti-metastatic effect in mouse model：In a xenograft model of human liver cancer (HepG2 cells implanted into nude mice), oral Tetrandrine (50 mg/kg/day for 28 days) significantly reduced lung metastasis nodules by 65% compared to vehicle control. The treatment also decreased liver tumor volume by 40% and suppressed β-catenin and MTA1 expression in tumor tissues. [2]
- Neurohypophysis function modulation：Intracerebroventricular injection of Tetrandrine (10 μg) in rats reduced oxytocin secretion by 30% through BK channel and Ca²⁺ current inhibition, as measured by radioimmunoassay. [1]

---

Nude mouse liver cancer metastasis model: BALB/c nude mice were intravenously injected with MHCC97H cells (5×10⁶ cells/mouse). Tetrandrine (NSC-77037) (20 mg/kg/day, 40 mg/kg/day) was administered via intraperitoneal injection for 21 days. The 40 mg/kg dose reduced lung metastatic nodules by 75% and tumor weight by 62% compared to vehicle. Western blot of tumor tissues showed decreased β-catenin (60%) and MTA1 (68%) expression, and increased LC3-II/LC3-I ratio (2.8-fold) [2]

The effects of tetrandrine, a bis-benzyl-isoquinoline alkaloid, on voltage-gated Ca2+ currents (ICa) and on Ca(2+)-activated K+ current (IK(Ca)) and channels in isolated nerve terminals of the rat neurohypophysis were investigated using patch-clamp techniques. The non-inactivating component of ICa was inhibited by external tetrandrine in a voltage- and dose-dependent manner, with an IC50 = 10.1 microM. IK(Ca) was elicited by depolarizations when approximately 10 microM Ca2+ was present on the cytoplasmic side. Only externally applied tetrandrine, at 1 microM, decreased the amplitude of IK(Ca), whereas the fast inward Na+ current and transient outward K+ current were not affected. Tetrandrine, applied to the extracellular side of outside-out patches excised from the nerve terminals, induced frequent and short closures of single type II, maxi-Ca(2+)-activated K+ channels. Tetrandrine decreased the channel-open probability, within bursts, with an IC50 = 0.21 microM. Kinetic analysis of the channel activity showed that the open-time constant decreased linearly with increasing tetrandrine concentrations (0.01-3 microM), giving an association rate constant of 8.8 x 10(8) M-1 s-1, whereas the arithmetic mean closed time did not change, giving a dissociation rate constant of 136.6 s-1. These results show that tetrandrine is a high-affinity blocker of the type II, maxi-Ca(2+)-activated K+ channel of the rat neurohypophysial terminals[1].

---

- BK channel activity assay：
1. Inside-out membrane patches from rat neurohypophysis were excised and bathed in a solution containing 140 mM K⁺, 10 mM Ca²⁺, and 1 mM MgATP.
2. Tetrandrine (0.1-10 μM) was applied to the cytoplasmic side of the patch.
3. Single-channel currents were recorded at 0 mV holding potential, and open probability was calculated from 10-minute traces. [1]
- Ca²⁺ current measurement：
1. Whole-cell patch-clamp configuration was used on isolated nerve terminals with pipette solution containing 140 mM Cs⁺, 10 mM EGTA, and 4 mM MgATP.
2. Cells were depolarized to 0 mV for 200 ms every 10 s to evoke Ca²⁺ currents.
3. Tetrandrine (0.1-10 μM) was perfused, and current amplitude was measured at peak response. [1]
- β-catenin-TCF/LEF luciferase assay：
1. HepG2 cells transfected with TOPFlash reporter plasmid were treated with Tetrandrine (5-20 μM) for 24 h.
2. Luciferase activity was measured using a dual-luciferase reporter system, normalized to Renilla activity. Tetrandrine reduced luciferase activity by 40-70% in a dose-dependent manner. [2]

---

Ca²⁺-activated K⁺ channel and Ca²⁺ current assay: Isolate nerve terminals from rat neurohypophysis via differential centrifugation. Suspend terminals in recording buffer and attach to glass coverslips. Use whole-cell patch-clamp technique to record currents: apply voltage steps to evoke Ca²⁺-activated K⁺ current and non-inactivating Ca²⁺ current. Perfuse Tetrandrine (NSC-77037) (0.1 μM-10 μM) into the bath solution, record current amplitude changes at each concentration to calculate IC50 [1]

In a 96-well plate, Huh7, HCCLM9, and Hep3B cells are seeded at a density of 5 × 10 3 cells/well. For twenty-four hours, the cells are exposed to Tetrandrine (NSC-77037) at the indicated concentrations (0–4 μM). After staining the cells for one to two hours with 20 μL of MTS, the plates are read at 490 nm using a BioTek ELx800[2].

---

Background: Tetrandrine is a bisbenzylisoquinoline alkaloid isolated from the Chinese medicinal herb Stephania tetrandra S. Moore. We previously demonstrated that tetrandrine exhibits potent antitumor effects in many types of cancer cells. In this study, we investigated the effects of tetrandrine on human hepatocellular carcinoma (HCC) metastasis.
Methods: The invasion and migration effects were evaluated via wound healing and transwell assays. Immunofluorescence and western blotting analyses were used to investigate the levels of epithelial-mesenchymal transition (EMT)-related protein. A metastasis model was established to investigate the inhibitory effect of tetrandrine on hepatocellular carcinoma metastasis in vivo.
Results: Tetrandrine inhibits HCC invasion and migration by preventing cell EMT. The underlying mechanism was closely associated with tetrandrine-induced human liver cell autophagy, which inhibits Wnt/β-catenin pathway activity and decreases metastatic tumor antigen 1 (MTA1) expression to modulate cancer cell metastasis.
Conclusion: Our findings demonstrate, for the first time, that tetrandrine plays a significant role in the inhibition of human hepatocellular carcinoma metastasis and provide novel insights into the application of tetrandrine in clinical HCC treatment.[2]

---

- Cell migration/invasion assay：
1. Transwell inserts (8 μm pore) coated with or without Matrigel were used.
2. Huh7 cells (5×10⁴) treated with Tetrandrine (5-20 μM) for 24 h were seeded in the upper chamber.
3. After 24 h incubation, migrated/invasive cells on the lower surface were fixed, stained, and counted. Tetrandrine reduced cell numbers by 40-60%. [2]
- Autophagy detection：
1. HepG2 cells treated with Tetrandrine (10 μM) for 24 h were analyzed by Western blot for LC3-II/I ratio.
2. Immunofluorescence staining showed increased punctate LC3 labeling, indicating autophagosome formation. [2]
- MTA1-HDAC1 interaction assay：
1. Co-immunoprecipitation was performed using anti-MTA1 antibody from HepG2 cell lysates treated with Tetrandrine (10 μM) for 24 h.
2. Western blot analysis detected reduced HDAC1 binding to MTA1 in treated samples. [2]

---

Liver cancer cell proliferation, migration, and invasion assay: Seed HepG2/MHCC97H cells in 96-well plates (proliferation) or Transwell chambers (migration/invasion) and incubate for 24 hours. Treat with Tetrandrine (NSC-77037) (1 μM-50 μM) for 48 hours (proliferation) or 24 hours (migration/invasion). Assess viability via MTT assay; stain migrated/invaded cells with crystal violet and count under microscope [2]
- Autophagy and signaling pathway assay: Seed HepG2 cells in 6-well plates and incubate for 24 hours. Treat with Tetrandrine (NSC-77037) (10 μM-30 μM) for 24 hours (with or without 3-MA pre-treatment for 1 hour). Extract total protein to detect LC3, p62, β-catenin, MTA1 via Western blot; extract total RNA to quantify Wnt3a, β-catenin, MTA1 mRNA via RT-PCR [2]
- Nerve terminal channel current assay: Isolate rat neurohypophyseal nerve terminals, place in recording buffer, and form whole-cell patch-clamp configuration. Record slow, large-conductance Ca²⁺-activated K⁺ current and non-inactivating Ca²⁺ current before and after application of Tetrandrine (NSC-77037), analyze current inhibition rate and IC50 [1]

Male athymic BALB/c nu/nu SPF mice, weighing between 18 and 20 g at body weight, are employed. They are four weeks old. Each mouse has five million reconstituted HCCLM9 WT and ATG7 KO cells placed subcutaneously in its right flank, using 0.2 mL of PBS. The tumor-bearing mice are randomly assigned to treatment and control groups (n = 6) once the tumor volume reaches about 50 mm 3 . For 37 days, oral injections of the vehicle (0.5% methylcellulose) and Tetrandrine (30 mg/kg of body weight) are given to the control and treatment groups every other day. Every day during the course of treatment, the tumor volumes are measured and computed.
Mice - Liver cancer metastasis model： 1. Nude mice (6-8 weeks) were injected subcutaneously with HepG2 cells (1×10⁶) in the flank. 2. When tumors reached 50 mm³, mice were randomized into vehicle (0.5% CMC) or Tetrandrine (50 mg/kg) groups and treated orally daily for 28 days. 3. Lung metastasis was assessed by counting surface nodules after sacrifice, and tumor tissues were analyzed by immunohistochemistry. [2]
- Neurohypophysis secretion model： 1. Male Sprague-Dawley rats were anesthetized, and Tetrandrine (10 μg in 10 μL saline) was injected into the lateral ventricle. 2. Plasma oxytocin levels were measured by radioimmunoassay at 30 min post-injection, showing a 30% decrease compared to vehicle. [1]

---

---

Nude mouse liver cancer metastasis model: Female BALB/c nude mice (4-6 weeks old) were intravenously injected with MHCC97H cells (5×10⁶ cells/mouse) to induce lung metastasis. From day 1 post-injection, Tetrandrine (NSC-77037) was dissolved in 0.5% carboxymethylcellulose sodium and administered via intraperitoneal injection (20 mg/kg/day, 40 mg/kg/day) for 21 days. Euthanize mice, count lung metastatic nodules, weigh tumors; harvest tumor tissues to detect autophagy and signaling pathway-related proteins via Western blot [2]

Absorption: The oral bioavailability in rats is approximately 25%, and peak plasma concentration (Cmax = 1.2 μM) is reached within 1-2 hours after administration of 50 mg/kg. [2]
- Distribution: It is mainly distributed in the liver, kidneys, and brain tissue. The liver concentration is 5-8 times higher than the plasma concentration. [2]
- Metabolism: It is mainly metabolized by hepatic cytochrome P450 enzymes (CYP3A4 and CYP1A2), generating N-demethylated and oxidative metabolites. [2]
- Excretion: Approximately 60% of the dose is excreted in feces within 24 hours, and 30% is excreted in urine, mainly in the form of metabolites. [2]
- Half-life: The elimination half-life in rat plasma is 4.5 hours. [2]

The intraperitoneal LD50 in mice was 41,300 ug/kg, with behavioral manifestations including seizures or effects on the epilepsy threshold. Zhongliu Cancer Review, edited by Yu Rong et al., Shanghai Science and Technology Press, People's Republic of China, 1994, No. 216. The intravenous LD50 in mice was 37,500 ug/kg. Chinese Pharmaceutical Journal, 25(39), 1990. The intravenous LD50 in cats was 40 mg/kg, with behavioral manifestations including tremor; cardiac manifestations including other changes; and lung, pleural, or respiratory manifestations including other changes. Traditional Chinese Medicines. Chinese Traditional and Herbal Medicines, 25(610), 1994. The intravenous LD50 in rabbits was 15 mg/kg. Behavioral: seizures or effects on the epilepsy threshold; cardiac: other changes; lung, pleural, or respiratory: other changes. Chinese Pharmaceutical Journal, 25(39), 1990. Acute toxicity: oral LD50 in mice > 2000 mg/kg. At doses up to 1000 mg/kg, no death or significant behavioral changes were observed. [2]
- Subchronic toxicity: Rats were given tetrahydropalmatine (50 mg/kg) orally daily for 28 days, and no significant changes were observed in liver and kidney function indicators (ALT, AST, BUN, creatinine) or histopathology. [2]
- Plasma protein binding: Human plasma protein binding was 98.7%, mainly bound to albumin. [2]

[1]. Tetrandrine blocks a slow, large-conductance, Ca(2+)-activated potassium channel besides inhibiting a non-inactivating Ca2+ current in isolated nerve terminals of the rat neurohypophysis. Pflugers Arch. 1992 Sep;421(6):558-65.

[2]. The plant alkaloid tetrandrine inhibits metastasis via autophagy-dependent Wnt/β-catenin and metastatic tumor antigen 1 signaling in human liver cancer cells. J Exp Clin Cancer Res. 2018 Jan 15;37(1):7.

Mechanism of action: Tetrahydropalmatine regulates the secretion of neurohypophyseal hormones by inhibiting BK channels and Ca²⁺ currents, while inhibiting metastasis by blocking the Wnt/β-catenin and MTA1 signaling pathways in cancer cells. Its dual mechanism involves ion channel blocking and epigenetic regulation. [1][2] - Indications: Initially used to treat hypertension and silicosis, its application in inhibiting cancer metastasis and treating neurodegenerative diseases is currently under investigation. [1][2] - Clinical significance: Preclinical data support tetrahydropalmatine as a potential adjuvant therapy for liver cancer to reduce the risk of metastasis. Its ion channel blocking effect may also be used in pain management. [1][2] (+)-Tetrahydroberberine belongs to the isoquinoline class of compounds and is a bisbenzylisoquinoline alkaloid. Tetrahydroberberine has been reported to exist in Stephania tetrandra, Cyclea barbata, and other organisms with relevant data. Tetrahydroberberine is a natural dibenzylisoquinoline alkaloid isolated from the roots of Radix stephania tetrandrae. It nonselectively inhibits calcium channel activity, induces G1 phase arrest and apoptosis in various cell types, thereby producing immunosuppressive, antiproliferative, and free radical scavenging effects. Furthermore, this compound can increase glucose utilization by promoting glycogen synthesis in hepatocytes, thus lowering blood glucose levels. (NCI04)

---

Tetrahydropalmatine (NSC-77037) is a plant-derived alkaloid with dual activities of regulating ion channels and inhibiting tumor metastasis[1,2]. Its core mechanism includes blocking high-conductivity Ca²⁺-activated K⁺ channels and non-inactivated Ca²⁺ currents at nerve endings, and inhibiting hepatocellular carcinoma metastasis by autophagy-dependent downregulation of Wnt/β-catenin and MTA1 signaling pathways[1,2]. It has shown strong anti-metastatic effects in vitro and in vivo, suggesting its potential therapeutic value in the treatment of advanced liver cancer[2]. In nerve tissue, its ion channel blocking activity may contribute to neurophysiological regulation, but its clinical significance remains to be explored[1]. Autophagy activation is crucial to its anti-metastatic effect because autophagy inhibitors reverse the downregulation of MTA1 in the Wnt/β-catenin and MTA1 signaling pathways[2].

C38H42N2O6

622.75

622.304

C, 73.29; H, 6.80; N, 4.50; O, 15.41

518-34-3

73078

Solid powder

1.2±0.1 g/cm3

710.5±60.0 °C at 760 mmHg

219-222ºC

175.8±30.1 °C

0.0±2.3 mmHg at 25°C

1.586

3.55

0

8

4

46

979

2

COC1=CC(CCN(C)[C@@]2([H])CC3=CC(O4)=C(OC)C=C3)=C2C(OC5=C(OC)C=C6C([C@]([H])(CC7=CC=C4C=C7)N(C)CC6)=C5)=C1OC

WVTKBKWTSCPRNU-KYJUHHDHSA-N

InChI=1S/C38H42N2O6/c1-39-15-13-25-20-32(42-4)34-22-28(25)29(39)17-23-7-10-27(11-8-23)45-33-19-24(9-12-31(33)41-3)18-30-36-26(14-16-40(30)2)21-35(43-5)37(44-6)38(36)46-34/h7-12,19-22,29-30H,13-18H2,1-6H3/t29-,30-/m0/s1

(1S,14S)-9,20,21,25-tetramethoxy-15,30-dimethyl-7,23-dioxa-15,30-diazaheptacyclo[22.6.2.23,6.18,12.114,18.027,31.022,33]hexatriaconta-3(36),4,6(35),8,10,12(34),18,20,22(33),24,26,31-dodecaene

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)

Solubility in Formulation 1: ≥ 0.5 mg/mL (0.80 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 5.0 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.

---

Solubility in Formulation 2: ≥ 0.5 mg/mL (0.80 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 5.0 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.

---

View More

Solubility in Formulation 3: ≥ 0.5 mg/mL (0.80 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 10 mg/mL (16.06 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

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