Simple Body Stretching Reduces Tumor Size By More Than 50%

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I’ve just recently added in passive hanging to my daily routine. A couple times a day. Feels good on my back, shoulders and spine

 

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Selective killing of transformed cells by mechanical stretch​

Abstract​

Cancer cells differ from normal cells in several important features like anchorage independence, Warburg effect and mechanosensing. Further, in recent studies, they respond aberrantly to external mechanical distortion. Consistent with altered mechano-responsiveness, we find that cyclic stretching of tumor cells from many different tissues reduces growth rate and causes apoptosis on soft surfaces. Surprisingly, normal cells behave similarly when transformed by depletion of the rigidity sensor protein (Tropomyosin 2.1). Restoration of rigidity sensing in tumor cells promotes rigidity dependent mechanical behavior, i.e. cyclic stretching enhances growth and reduces apoptosis on soft surfaces. The mechanism of mechanical apoptosis (mechanoptosis) of transformed cells involves calcium influx through the mechanosensitive channel, Piezo1 that activates calpain 2 dependent apoptosis through the BAX molecule and subsequent mitochondrial activation of caspase 3 on both fibronetin and collagen matrices. Thus, it is possible to selectively kill tumor cells by mechanical perturbations, while stimulating the growth of normal cells.

Introduction​

The transformed phenotype was initially described in early studies of cancer cell growth, since tumor cells would often grow on soft agar plates, while normal cells from the same tissue required a rigid surface for growth [1]. Recent studies have shown that cancer cells from many different tissues lack rigidity sensors, which could explain the growth on both soft and rigid matrices. Restoration of rigidity sensing, through cytoskeletal protein expression in cancer cells, blocks transformed growth [2,3]. Further, normal cells can grow on soft surfaces after depletion of cytoskeletal proteins that are required for rigidity-sensing. Because these transitions will occur in cells from many different tissue backgrounds, it appears that the transformed phenotype is a major cell state that is related to the activation of adult cell growth for wound repair in many different tissues [4]. This raises many questions about the transformed cell state and how it may be fundamentally different from the normal cell state. For example, the morphology and mechanical properties of the cancer cells are dramatically different from the normal cells, which raises the possibility that there may be substantial differences in the mechanosensitivity between normal cells and cancer cells.

Several reports have indicated that cancer cell growth is vulnerable to mechanical stresses. Fluid shear-induced killing of the circulating tumor cells and adherent cancer cells can be explained by increased sensitivity to mechanical stresses [5,6]. In addition, ultrasonic and shock-wave therapies have demonstrated that cancer cells are particularly sensitive, although concerns were raised about increased metastasis and healthy tissue damage [[7], [8], [9], [10]]. Despite many possible explanations, studies showing inhibition of tumor growth after stretching or exercise in mice models are consistent with mechanical stress-dependent tumor growth inhibition [11,12]. The mechanosensitivity could be tumor specific or it could possibly be a general property of the transformed state [4].

Because the presence or absence of a rigidity-sensing complex correlates strongly with the normal (rigidity-dependent) and transformed growth (rigidity-independent) states, respectively, it is relevant to understand the important elements of the rigidity sensors. They are sarcomeric units of ~2 μm in length with anti-parallel actin filaments anchored to the matrix adhesion sites that transiently assemble to measure rigidity. Myosin IIA contraction pulls the adhesions to a constant displacement of about 100 nm for 30 s irrespective of substrate rigidity and can generate very large forces per myosin head [2,13]. If the contractile force exceeds ~25 pN, then the matrix is considered rigid and the adhesions will be reinforced. Adhesion disassembly and eventual cell apoptosis will occur if the surface is soft. A common mechanism of malignant transformation is the depletion of Tpm2.1 or the increased expression of Tpm3 [14,15]. Decreasing the ratio of Tpm2.1 to Tpm3 causes the loss of matrix rigidity sensing and enables transformed cell growth. This process can occur even in the normal cells [2,3]. In contrast, restoration of Tpm2.1 level in many cancer cells re-establishes rigidity-dependent growth and inhibits transformation in those cells [3]. Other mechanosensory cytoskeletal proteins (myosin IIA, α-actinin, filamin A, AXL, ROR) are part of the rigidity sensing complex. Depletion of these sensory proteins and other proteins like scaffolding proteins (caveolin1) promotes cell transformation, while their restoration in cancer cells can block transformed cell growth [2,3,16,17]. Thus, the transformed state of cancer cells appears in many different tissue backgrounds to result from the loss of rigidity sensing, i.e. transformed cells grow on soft surfaces because they fail to sense the surface softness.

There are a number of common features of cancer cell mechanics that differ from normal cells, including a decrease in the rigidity of the cortical cytoskeleton [16,18] and increased traction forces on matrices [19]. In addition, there are multiple reports demonstrating the altered expression and function of calcium channels as well as increased calpain activity in cancer cells [20,21]. In this study, we examine the mechanical stresses that cause selective apoptosis of the transformed cells, but don't damage the normal cells. Particularly, we observe that physiologically relevant cyclic mechanical stretch inhibits transformed cell growth and activates apoptosis. Surprisingly, restoration of the matrix rigidity sensing in transformed cells increases cell growth and survival upon cyclic stretch, particularly on soft surfaces. The mechanism of mechanical apoptosis, mechanoptosis, involves the mechanosensitive channel Piezo1-dependent calcium influx that activates calpain2-mediated mitochondrial apoptosis. Thus, mechanical sensitivity is a common feature of many transformed cells that has important implications for inhibiting tumor growth.

Discussion​

These studies show that normal and transformed cells from many different tissues react in opposite ways to the mechanical perturbation of stretch, particularly on soft surfaces. For many different cancer cell lines, the restoration of rigidity sensing causes rigidity-dependent growth, while depletion of rigidity sensors in normal cells results in transformed growth despite different tissue backgrounds [3,4]. In transformed cells, cyclic stretch initially causes cell elongation on rigid surfaces that is dependent on the stretch frequency as well as the magnitude of strain. The frequency and magnitude of stretch used in these studies are chosen to be in a physiologically relevant range for exercise. Prolonged periods of cyclic stretch inhibit transformed cell growth and increase apoptosis, particularly on soft surfaces. In contrast, cyclic stretch promotes normal cell growth and inhibits apoptosis on soft surfaces. Further, the stretch-induced apoptosis is calpain dependent (primarily calpain 2) and calpain acts downstream of stretch-mediated calcium influx to activate BAX molecule to initiate mitochondrial apoptosis. The mechanosensitive Piezo1 calcium channels are needed for calcium influx in the background of the transformed cell state.

Numerous studies of the transformed cell state indicate that normal cells from a variety of different tissues can be transformed in a process of mimicking the activation of growth in wound healing [3,4]. Thus, tumor cell growth has been described as prolonged wound healing [44]. Further, a common feature of wound healing in a variety of tissues is the overexpression of miR-21 that is correlated with cancer severity in many cases [45]. Of the several miR-21 targets that are involved in tumor suppression, the depletion of Tpm2.1 correlates best with tumor severity [46,47] and analysis of the TCGA database reveals that TPM2 expression (encodes Tpm2.1) levels are significantly lower in tumor cells from 18 different tissues than in their matched normal tissues (Supplementary Fig. 19). From, earlier studies of the transformed phenotype, transformation correlates with the loss of rigidity sensing and a change in the cortical actin organization that makes cancer cells softer [2,16]. However, the traction forces that most cancer cells exert on matrices are higher than their normal counterparts indicating greater myosin-IIA contractility generation in cancer cells. This observation is in line with multiple studies emphasizing the importance of RhoA-mediated myosin IIA contractility in cell transformation, tumorigenesis and metastasis [48–53]. Interestingly, restoration of rigidity sensing in cancer cells causes a decrease in traction forces.2, 3 Thus, it is logical to consider the ‘transformed state’ as a distinct cell state that can occur in cells from different tissues with different expression patterns [4]. Further, cells from different tissues can be toggled between the transformed and the normal state by changing the level of expression of several cytoskeletal proteins to block or enable, respectively, matrix rigidity sensing [3].

A relatively common feature of cancer cells is that they have elevated levels of calcium channels and calpain proteases [20,21]. Higher levels of mechanosensitive calcium channels and their altered behavior could explain the increased susceptibility of cancer cells to stretch-induced apoptosis. However, the transformation of fibroblasts and epithelial
cells with Tpm2.1 KD will also sensitize those cells to stretch-induced apoptosis. RNAseq analyses of cells with and without rigidity sensing due to presence or absence of Tpm2.1 expression show that altering the level of only Tpm2.1 expression will result in significant changes in expression of about thousand proteins [3]. This indicates that transformed cells are significantly different from their normal counterparts even if depletion of single proteins can cause transformation. Thus, we suggest that transformed cells, whether from a tumor or from a normal cell background, will undergo apoptosis in response to the appropriate mechanical forces that can be mimicked by cyclic stretch.

There are previous reports of mechanical force-induced cancer cell death. Continuous flow forces can cause the apoptosis of several different cancer cells attached to surfaces, whereas oscillatory flow forces do not [6]. The apoptotic pathway involves bone morphogenetic protein receptor, Smad1/5, and p38 MAPK unlike our findings. Another
study shows that high shear forces on suspended circulating tumor cells initiates apoptosis possibly through an oxidative stress-induced mitochondrial apoptotic pathway [5]. Further, recent findings reveal that the stretching of mice for 10 min a day for four weeks suppresses breast cancer tumor growth by 50% compared to the non-stretched controls [11]. From these and other studies, there are strong indications that cancer cells may be mechanically vulnerable and thus sensitive to mechanical forces. Here we show that the increased mechanical sensitivity is linked to the transformed cell state and not to a specific tissue or cell type. Thus, it seems that this feature could be exploited to damage many different types of tumor cells, particularly in a metastatic state where cells may not be protected by tumor fibrosis.

In Fig. 8, we present a working model of mechanical stretch-induced transformed cell killing, which we are describing as ‘mechanoptosis’. Prolonged application of mechanical stresses on the transformed cells triggers a rise in calcium levels through activation of Piezo1 channels. The rise in intracellular calcium level causes activation of calpain 2 protease which initiates a mitochondrial apoptotic pathway through its downstream effector, the BAX molecule. BAX translocation to mitochondria disrupts outer membrane permeability to leak mitochondrial cytochrome c into cytosol. Eventually cytochrome c activates caspase proteases through series of biochemical reaction to trigger apoptosis. In contrast, mechanical stretching of normal cells does not cause a rise in calcium levels because of a more tightly regulated calcium homeostatic mechanism. Thus, cyclic stretch promotes the normal cell growth and survival especially on the soft surfaces, whereas it promotes apoptosis of transformed cells.

There are several studies that indicate cancer cells are sensitive to calcium-dependent apoptosis. For example, prolonged exposure to the anticancer agent, FPDHP promoted apoptosis in several human cancer cells via a calpain-mediated mitochondrial apoptotic pathway [54]. Similarly, it was observed that capsaicin in combination with camptothecin enhanced the apoptosis of small cell lung carcinoma cells and it was mediated by elevation of intracellular calcium and calpain activation [55]. In hepatocellular carcinoma cells, TNFα-mediated calcium influx enhanced the apoptosis through activating calpain/IAP/caspase3 pathway [56]. In another study, lenalidomide accelerated the apoptosis of myeloid leukemia cells by increasing cytosolic calcium level via GPR68 calcium channel activation and subsequent calpain 1 activation [57]. All together, these studies affirmed the central role played by calcium influx-mediated calpain pathway in initiating cancer cell apoptosis in response to the different biological/chemical activators.

Although most cancer cells are transformed, there are many other aspects that are important for tumor growth [58–60]. We suggest that transformation is necessary for tumor growth because the normal microenvironment sensory system for matrix rigidity is depleted but it is far from sufficient for life-threatening tumor growth. Hundreds of other proteins are also altered beyond those lost with rigidity sensor depletion in tumor cells, making it difficult to link the increased mechanical sensitivity to a single factor. Despite the fact that we do not fully understand why transformed cells are more sensitive to calcium loading with cyclic stretch, that sensitivity can provide new approaches for killing cancer cells while actually stimulating the normal cell growth. Since many of the transformed cells that were used in these studies are not tumorigenic but could be considered as pre-tumorigenic, transformation is a likely prerequisite for tumor formation. Thus, we suggest that even early-stage tumor cells would be sensitive to mechanoptosis. Much more work is needed to determine if this feature can be exploited in an in vivo setting to damage cancer cells.