HTRA1 & PEMF

This post examines Tai 2024, a QuantumTx group publication that is a follow up to a previous study showing that their brand of PEMF elicits the release of the High Temperature Requirement A1 serine protease that might mitigate the progression of cancer. Both myoblasts and myotubules were tested in this study. The direction of PEMF relative to the force of gravity was also examined. These conditioned media were used for cancer cell line based tests of anti-carcinogenicity.

The featured image is meant to illustrate the complexity of what this might mean in real muscle. See the PEMF and breast cancer post. This particular post will simply attempt to summarize the background study.

HTRA1 background

A Master’s thesis on the many roles of HTRA1, positive and negative would not help the understanding of a very interesting arsenal in the treatment of breast cancer by QuantumTx.

HTRA1, links to some mechanism cartoons

For impatient visual learners, these are some cartoons that give an overview of different ways HTRA1 may affect cancer. Song 2024, see the reference section, is perhaps the best background for this post.

• Li 2019 Fig 1. ⭐ gets a star as a sequence alignment of the HTRA1 protein sequences in different species. Tai 2024 comment on species differences.
• Oka 2020 Secreted HTRA1 acts as a protease on transforming growth factor (TGF-β), canonical Wingless/Integrated (WNT) and NOTCH signaling pathways. This review cartoon has intracellular HTRA1 binding to β-catenin suggesting that the HTRA1 that is not released cold be important.
• Chen 2021shows a few cancer pathways inhibited by HTRA1.
• Liu 2024 HTRA1 is up regulated in some forms of colorectal cancer. HTRA1 could interact with the glutamate/cystine transporterSLC7A11 and up-regulate the expression of SLC7A11, which in turn makes the CRC less susceptible to death by oxidative stress mechanisms. Cystine is a precursor of glutathione synthesis.
• Song 2024 Fig 1 highlights the confusing biology of driving with one foot on the Notch 3 matrix metallo protease (mmp) brake and the other foot on the vascular smooth muscle protein synthesis TGF beta accelerator. HTRA1 inhibits both.
• Song 2024 Fig 2 is a complicated cartoon of the multifaceted role of HTRA1 in the eye vascular disease wet AMD Age related Macular Degeneration.
• Song 2024 Fig 3 illustrates the complicated nature of HTRA1 in the tumor mcroenvironment.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL)

CARASIL is caused by mutations in the HTRA1 gene which reduces the activity of this protease or a protease with no activity at all. Mutant HTRA1 is unable to suppress TGF-β activity. This leads to damage to small blood vessels in the brain.

a genomic data analysis of breast cancers and HTRA1, Zhao 2023

“HTRA1 expression is associated with immune-cell infiltration, response to chemotherapy, and survival outcomes in BRCA. HTRA1 has the potential to serve as a promising biomarker and therapeutic target moving forward.”

Where is HTRA1 expressed?

A previous post on this site focused on the influence of PEMF on fat and muscle. Note that HTRA1 is highly expressed in fat and gall bladder, of all places. Note that there is no skeletal muscle source in the bar graph from ProteinAtlas.org. Fat is starred.

by tissue

by cell line

The ASAC52telo adipose cell line expresses HTRA1

HTRA1 released by muscle by PEMF, Tai 2024

Introduction

The following is a bullet point summary of the Tai 2024 introduction. The reader of this post is invited to consult this open access publication for the referenced peer reviewed publications.

• Skeletal muscle may account for 40% of a human’s weight and that increased muscle mass is correlated with lower incident of certain cancers including prostate, breast, and colon.
• Exercise before, during, and after breast cancer therapy is associated with improved outcome in humans and animal models.
• Myokines released from muscle are thought to be part of the benefit.
• Myokines are a collection of cytokines and other bioactive agents that are released into the interstitial fluid and bloodstream, interact with muscle and other tissues to modulate their metabolism and inflammatory status. The featured image in the top bar shows an H&E stained section of a muscle fiber with a capillary.
• Some myokines inhibit the epithelial-mesenchymal (EMT) transition, migration, and invasion.
• Myokines mitigate cancer-permissive comorbidities such as obesity, low-grade inflammation.
• PEMF is intended to be a solution for those who are unable to exercise via PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator 1alpha) transcriptional pathway which naturally involves mitochondria.
• High-Temperature Requirement A1 (HTRA1) is a candidate anticancer myokine released into PEMF-induced conditioned media.
• HTRA1 levels are downregulated in several forms of cancer and correlate with increased tumor growth, epithelial-mesenchymal transition (EMT), metastasis, and chemoresistance.
• Decreased levels of HTRA1 are correlated with more aggressive forms of breast cancers.
• HTRA1 is predominantly secreted into the bloodstream as a cleavage product of the full-length protein, conferring upon it paracrine action on adjacent and distal tissues.
• HTRA1 induction with exercise has yet to be convincingly shown. HTRA1 expression has been shown to be upregulated upon magnetic exposure of muscle cells.
• The neighborhood of the human HTRA1 gene was investigated independently of Tai 2024

• HTRA1 was found to be in the neighborhood of the fibroblast growth factor receptor receptor 2 FGFR2 and DMBT1, deleted in malignant brain tumors 1.
• TACC2 is thought to be associated with the progression of breast cancer. ATE1 codes for arginyl transferase, which is is involved in posttranslational conjugation of arginine to N-terminal aspartate or glutamate residues. Conjugation of arginine to the N-terminal aspartate or glutamate targets proteins for ubiquitin-dependent degradation.

Basic protocol

• C2C12 muscle cells were exposed for 10 minutes with 1.5 mT, 6 msec bursts of 7 kHz at a repetition of 15 Hz PEMF.
• Differentiated myotubes in serum-free DMEM were subjected to 1.5 mT PEMF for 10 min and allowed to repose for 6 h or 24 h in a standard cell culture incubator. Control cells were placed in the PEMF device without powering. Conditioned media was collected after centrifugation at 1200 rpm for 5 min. The significance is that carcinogenicity tests will be performed in absence of growth factors normally found in growth serum.
• Breast cancer cells, 24 h post seeding, were given PEMF muscle cell conditioned medium.
• Breast caner cell tumors were also grown in fertilized chicken eggs to test the ability of the conditioned media to inhibit vascularization of the cultured tumor cells..

Results, by figure

Fig 1 PEMF conditioned media from myotubules inhibits breast cancer cells

Myotubule conditioned media was introduced to a variety of breast cancer cell lines to test the hypothesis that the myotubules release something that inhibits carcinogenicity as measured by a variety of in vitro assays.

Fig 2 The same PEMF conditioned media inhibits vascularization of human breast cancer cells in a chicken embryo model

MCF-7 breast cancer cells were injected into fertilized chicken eggs and allowed to grow for six days. Co-injection of control and PEMF conditions media tested the ability of myokines to prevent vascularization as the cells grew into tumor like clumps. Something in the conditioned medium prevented vascularization.

Fig 3 Exercised mouse conditioned media produces the same anti-cancer results

Tai and coauthors used serum from sedentary mice, exercising mice, and mice exposed to PEMF without exercise. Sera from mice undergoing both treatments slightly, but significantly, reduced breast cancer cell line tumorigenicity as resumed by cell culture assays.

Fig 4 A complex paradigm involving myoblasts and myotubules, both exist in the same muscle

Please consult the featured figure. Myoblasts and myotubules exist together in mature muscle. Could their communication be part of the therapy? Myoblasts and myotubules were exposed to directional PEMF. Control and PEMF conditioned media from (cCM and pCM) were given to cultured myotubules. Then these conditioned media were given to breast cancer cell lines. Cell culture carcinogenicity assays were performed. A consistent theme of QuantumTx publications is that the magnetic field in the same direction as gravity is more effective in the release of myokines. The cartoons are pretty good in this figure and make a really complicated experimental protocol easier to follow. The cell memory of prior exposures will impact the model in which human patients receive a positive benefit from only 10 minute exposure per week. However, the greatest response was achieved with direct myotube exposure to downward PEMFs preceded by preconditioning with the downward field-generated pCM from myoblasts

Fig 5 extending Fig 4 paradigms to carcinogenicity assays

The secretomes generated from pCM (down) preconditioned myotubes, with or without direct myotube PEMF exposure inhibited invasion of a martrix, slowed growth back into a scratch, and slowed colony formation.

Fig 6 HTRA1 promotes preconditioning efficiency

Panels A, B PEMF exposed differentiated C2C12 myotubules, but not proliferative myoblasts, release HTRA1 into the medium. There’s not much change in the amount of HTRA1 inside the cells.

Panels 6C, D The authors examine the relationship between cell culture density and HTRA1 expression. High culture density tends to result in more HTRA1 release. Normal cells stop dividing when they contact one another. Silencing RNA (siRNA) was used to keep HTERA1 messenger RNA from being translated into protein. The anti-proliferative inhibitor of a cell cycle dependent protein kinase, p21, was decreased at the protein level by removing HTRA1 from the milieu. 40% less HTRA1 translates to 40% less p21. The decrease in protein levels of the transcription factor for muscle genes, myoD, was decreased by knocking down HTRA1.

Panel 6E (i) This is an example Western blot. The dark bands are proteins of interest. The strategy is to determine if PEM-ing myoblasts and then using the condition medium to stimulate differentiated myotubules… Tai 2024 were using a basal medium without serum with growth factors and such.

Panel 6E (ii) Two rounds of down PEMF are best for increasing protein levels of the muscle transcription factor myoD.

Panel 6E(iii) Myogenin is another muscle specific transcription factor that was optimally increased by two rounds of PEMF on myoblasts and myotubules.

Panel 6E (iv) Desmin is an intermediate filament protein. Filaments of desmin provide structure to the myocyte but do not participate in contraction as actin and myosin filaments do. Twice down PEMFed preps shown an increase in desmin over myotubules not treated with conditioned medium from PEMFed myoblasts. See the featured image. Myoblasts may be in close proximity to myotubules and mature fibers.

Panel 6E (v) PEMF preconditioned media promotes more PEMF release with the next PEMF session.

Panel 6G In a totally different experiment, spiking MCF7 breast cancer cells with recombinant HTRA1 decreases the live count. Recombinant generally refers to proteins engineered to be expressed in bacteria.

Panel 6H What happens when antibodies are used to “immunoprecipitate HTRA1 from the serum? Do we still see the same benefit from the PEMF conditioning? The answer is no. As a control, the authors tossed in some non-specific antibody just to show that the lack of effect was not due to the antibody, not the HTRA1 removal.

Panel 6I Does exercise plus PEMF increase HTRA1 in mice? Both increase HTRA1 in mouse serum over the control, but no synergy was observed in these experiments.

Discussion

This post takes concepts from the Discussion section as it was written back in 2024. The author of this post went back and asked “What induces the transcription of the HTRA1 gene” and came back with a mito hormesis answer.

Tai 2024 discussion

• Exercise improves the cancer prognosis, but not everyone can exercise including the frail.
• Aminoglycoside antibiotics that block the TRPC1 Ca²⁺ channel. Conductance through this channel is required for secretion of myokines.
• There might be some breast cancer cell line/serum effects that are species dependent to be addressed in future experiments.
• There might also be changes in response of cells to PEMF dependent on whether they are in suspension or attached to a surface.
• The epithelial–mesenchymal transition (EMT) was briefly commented on.
• Vascularization in the chick embryo model was briefly commented on.
• Release of anti-cancer agents from cells and whole animals was discussed. It should be added that the whole mice were exposed. In a subsequent study, just the thigh, fat, skin and bones of human volunteers was exposed. See PEMF and breast cancer post.
• PEMF increases expression of HTRA1 in C2C12 muscle cells.
• Muscle health is important for cancer survival. Attenuating TGF-β signaling required for angiogenesis may be a mechanism of HTRA1 released from muscle.
• Muscle is not known as a major producer of HTA, The HTRA1 gene is in the neighborhood. Perhaps there is a connection. (post author’s thought)
• Tai 2024 did not rule out other myokines and secreted factors as part of the PEMF response.

He 2022 an H₂O₂, mito hormesis link

This finally comment is from the post author’s attempt to find a mitohoremesis link to HTRA1. He 2022 did not identify the transcription factor but they did link H₂O₂ to increased gene

What was known before

• Both environmental and genetic risk factors are involved in Adult Macular Degeneration AMD pathogenesis.
• The HTRA1 gene has been shown to be one of the major causal genes to AMD.
• SNP rs11200638 in HtrA1 promoter site was deemed as the AMD-associated allele primarily based on the assumption that SNP rs11200638 up-regulated HTRA1 expression.

What this study adds

• In the present study, diverse doses of H₂O₂ and LPS were tested for the induction of oxidative and inflammatory reactions. Adult retinal pigment epithelial cells produce superoxide dismutase 1 in response to H₂O₂ and lipopolysaccharide and the protein level Fig 1 and transcript level Fig 2. 200 μM H₂O₂ increased both about 2-3 fold.
• The authors that the found variant rs11200638 upregulated the responsiveness of the HTRAA1 promoter after H₂O₂.
• The increased HtrA1 expression induces RPE cell apoptosis and growth inhibition.

He 2022 is a very nice study that this post will not get into. The nice thing about He2022 is that it ties HTRA1 production by PEMF to other work by the QuantumTx group.

References

• He F, Li X, Cai S, Lu L, Zhang T, Yang M, Fan N, Wang X, Liu X. Polymorphism rs11200638 enhanced HtrA1 responsiveness and expression are associated with age-related macular degeneration. Eye (Lond). 2022 Aug;36(8):1631-1638. PMC free paper
• Song S, Li X, Xue X, Dong W, Li C. (2024) Progress in the Study of the Role and Mechanism of HTRA1 in Diseases Related to Vascular Abnormalities. Int J Gen Med. 2024 Apr 18;17:1479-1491. PMC free paper.
• Tai YK, Iversen JN, Chan KKW, Fong CHH, Abdul Razar RB, Ramanan S, Yap LYJ, Yin JN, Toh SJ, Wong CJK, Koh PFA, Huang RYJ, Franco-Obregón A. (2024) Secretome from Magnetically Stimulated Muscle Exhibits Anticancer Potency: Novel Preconditioning Methodology Highlighting HTRA1 Action. Cells. 2024 Mar 5;13(5):460. PMC free paper
• Wong C.J.K., Tai Y.K., Yap J.L.Y., Fong C.H.H., Loo L.S.W., Kukumberg M., Frohlich J., Zhang S., Li J.Z., Wang J.W., et al. Brief exposure to directionally-specific pulsed electromagnetic fields stimulates extracellular vesicle release and is antagonized by streptomycin: A potential regenerative medicine and food industry paradigm. Biomaterials. 2022 free paper
• Zhao D, Li W, Wang Y, Zhang G, Bai X, Yu H. HTRA1 expression is associated with immune-cell infiltration and survival in breast cancer. Transl Cancer Res. 2023 Dec 31;12(12):3503-3521. doi: 10.21037/tcr-23-773. Epub 2023 Dec 19. PMC free paper